Heteroaryl compounds and uses thereof

ABSTRACT

The present invention provides compounds, pharmaceutically acceptable compositions thereof, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 12/426,495, filed Apr. 20, 2009, which is a continuation-in-part ofU.S. application Ser. No. 12/253,424, filed Oct. 17, 2008, which claimspriority to U.S. provisional application Ser. No. 60/981,432, filed Oct.19, 2007, and U.S. provisional application Ser. No. 61/052,002, filedMay 9, 2008, the entirety of each of which is hereby incorporated hereinby reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors ofprotein kinases. The invention also provides pharmaceutically acceptablecompositions comprising compounds of the present invention and methodsof using said compositions in the treatment of various disorders.

BACKGROUND OF THE INVENTION

The search for new therapeutic agents has been greatly aided in recentyears by a better understanding of the structure of enzymes and otherbiomolecules associated with diseases. One important class of enzymesthat has been the subject of extensive study is protein kinases.

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within the cell. Protein kinases are thought tohave evolved from a common ancestral gene due to the conservation oftheir structure and catalytic function. Almost all kinases contain asimilar 250-300 amino acid catalytic domain. The kinases may becategorized into families by the substrates they phosphorylate (e.g.,protein-tyrosine, protein-serine/threonine, lipids, etc.).

In general, protein kinases mediate intracellular signaling by effectinga phosphoryl transfer from a nucleoside triphosphate to a proteinacceptor that is involved in a signaling pathway. These phosphorylationevents act as molecular on/off switches that can modulate or regulatethe target protein biological function. These phosphorylation events areultimately triggered in response to a variety of extracellular and otherstimuli. Examples of such stimuli include environmental and chemicalstress signals (e.g., osmotic shock, heat shock, ultraviolet radiation,bacterial endotoxin, and H₂O₂), cytokines (e.g., interleukin-1 (IL-1)and tumor necrosis factor α (TNF-α)), and growth factors (e.g.,granulocyte macrophage-colony-stimulating factor (GM-CSF), andfibroblast growth factor (FGF)). An extracellular stimulus may affectone or more cellular responses related to cell growth, migration,differentiation, secretion of hormones, activation of transcriptionfactors, muscle contraction, glucose metabolism, control of proteinsynthesis, and regulation of the cell cycle.

Many diseases are associated with abnormal cellular responses triggeredby protein kinase-mediated events as described above. These diseasesinclude, but are not limited to, autoimmune diseases, inflammatorydiseases, bone diseases, metabolic diseases, neurological andneurodegenerative diseases, cancer, cardiovascular diseases, allergiesand asthma, Alzheimer's disease, and hormone-related diseases.Accordingly, there remains a need to find protein kinase inhibitorsuseful as therapeutic agents.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors of one or more protein kinases. Such compounds have thegeneral formula I:

or a pharmaceutically acceptable salt thereof, wherein Ring A, Ring B,m, R^(x), R^(y), W, and R¹ are as defined herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with abnormal cellular responsestriggered by protein kinase-mediated events. Such diseases, disorders,or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofkinases in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by such kinases; andthe comparative evaluation of new kinase inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the EGF inhibiting activity of compound I-1.

FIG. 2 depicts the results of compound I-1 in a “washout” experiment ascompared with compound I-93.

FIG. 3 depicts dose response inhibition of EGFR phosphorylation andp42/p44 Erk phosphorylation with compounds I-16 and I-17 in A431 cells.

FIG. 4 depicts dose response inhibition of EGFR phosphorylation andp42/p44 Erk phosphorylation with compound I-19 in A431 cells.

FIG. 5 depicts dose response inhibition of EGFR phosphorylation withcompound I-1 in A431 cells as compared with its “reversible control”compound (I^(R)-3).

FIG. 6 depicts the results of compound I-1 in a “washout” experiment ascompared with its “reversible control” compound (I^(R)-3).

FIG. 7 depicts MS analysis confirming covalent modification of ErbB4 bycompound I-1.

FIG. 8 depicts MS analysis confirming covalent modification of ErbB1 atCys797 by compound I-1.

FIG. 9 depicts the inhibition of BTK signaling in Ramos cells bycompound I-13.

FIG. 10 depicts the results of compound I-13 in a “washout” experimentwith BTK in Ramos cells.

FIG. 11 depicts MS analysis of the tryptic digests confirming covalentmodification of TEC kinase by compound I-13.

FIG. 12 depicts MS analysis confirming covalent modification of BTK bycompound I-63.

FIG. 13 depicts MS analysis confirming covalent modification of BTK bycompound I-66.

FIG. 14 depicts an amino acid sequence for BTK (SEQ ID 1).

FIG. 15 depicts an amino acid sequence for TEC (SEQ ID 2).

FIG. 16 depicts an amino acid sequence for ITK (SEQ ID 3).

FIG. 17 depicts an amino acid sequence for BMX (SEQ ID 4).

FIG. 18 depicts an amino acid sequence for JAK3 (SEQ ID 5).

FIG. 19 depicts an amino acid sequence for TXK (SEQ ID 6).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description ofCompounds of the Invention

In certain embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   Ring A is an optionally substituted group selected from phenyl, an    8-10 membered bicyclic partially unsaturated or aryl ring, a 5-6    membered monocyclic heteroaryl ring having 1-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, or an 8-10    membered bicyclic heteroaryl ring having 1-5 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   Ring B is phenyl, a 5-6 membered heteroaryl ring having 1-3    heteratoms independently selected from N, O or S, a 5-6 membered    saturated heterocyclic ring having 1-2 heteroatoms independently    selected from N, O or S, or an 8-10 membered bicyclic partially    unsaturated or aryl ring having 1-3 heteroatoms independently    selected from N, O or S;-   R¹ is a warhead group;-   R^(y) is hydrogen, halogen, CN, lower alkyl, or lower haloalkyl;-   W is a bivalent C₁₋₃ alkylene chain wherein one methylene unit of W    is optionally replaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—,    —N(R²)SO₂—, —SO₂N(R²)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or    —SO₂—;-   R² is hydrogen or optionally substituted C₁₋₆ aliphatic, or:    -   R² and a substituent on Ring A are taken together with their        intervening atoms to form a 4-6 membered saturated ring, or:    -   R² and R^(y) are taken together with their intervening atoms to        form a 4-7 membered carbocyclic ring;-   m is 0-4;-   each R^(x) is independently selected from —R, halogen, —OR, —CN,    —NO₂, —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂,    —NRSO₂R, or —N(R)₂; or:    -   R^(x) and R¹ are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group and 0-3 groups independently selected from        oxo, halogen, CN, or C₁₋₆ aliphatic; and-   each R group is independently hydrogen or an optionally substituted    group selected from C₁₋₆ aliphatic, phenyl, a 4-7 membered    heterocylic ring having 1-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl    ring having 1-4 heteroatoms independently selected from nitrogen,    oxygen, or sulfur.

In certain embodiments, the present invention provides a compound offormula II:

or a pharmaceutically acceptable salt thereof, wherein:

-   Ring A is an optionally substituted group selected from phenyl, an    8-10 membered bicyclic partially unsaturated or aryl ring, a 5-6    membered monocyclic heteroaryl ring having 1-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, or an 8-10    membered bicyclic heteroaryl ring having 1-5 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R¹ is a warhead group;-   R^(y) is hydrogen, halogen, CN, lower alkyl, or lower haloalkyl;-   G is CH, or N;-   W is —NR²—, —S—, or —O—;-   R² is hydrogen or optionally substituted C₁₋₆ aliphatic, or:    -   R² and a substituent on Ring A are taken together with their        intervening atoms to form a 4-6 membered saturated ring;-   m is 0-4;-   each R^(x) is independently selected from —R, halogen, —OR, —CN,    —NO₂, —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂,    —NRSO₂R, or —N(R)₂; or:    -   R^(x) and R¹ are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group and 0-3 groups independently selected from        oxo, halogen, CN, or C₁₋₆ aliphatic; and-   each R group is independently hydrogen or an optionally substituted    group selected from C₁₋₆ aliphatic, phenyl, a 4-7 membered    heterocylic ring having 1-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl    ring having 1-4 heteroatoms independently selected from nitrogen,    oxygen, or sulfur.

2. Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Suitable aliphatic groups include, but are not limited to, linear orbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₈ (or C₁₋₆) saturated orunsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalentcyclopropyl group of the following structure:

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. The term “aryl”may be used interchangeably with the term “aryl ring”. In certainembodiments of the present invention, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,anthracyl and the like, which may bear one or more substituents. Alsoincluded within the scope of the term “aryl”, as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-”, used alone or as part of alarger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring”, “heteroarylgroup”, or “heteroaromatic”, any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclicradical”, and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclicgroup”, “heterocyclic moiety”, and “heterocyclic radical”, are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(o); —(CH₂)₀₋₄OR^(o); —O(CH₂)₀₋₄R^(o), —O—(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄CH(OR^(o))₂; —(CH₂)₀₋₄SR^(o); —(CH₂)₀₋₄Ph, which may besubstituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(o); —CH═CHPh, which may be substituted with R^(o);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(o); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(o))₂; —(CH₂)₀₋₄N(R)C(O)R; —N(R^(o))C(S)R^(o);—(CH₂)₀₋₄N(R)C(O)NR^(o) ₂; —N(R^(o))C(S)NR^(o) ₂;—(CH₂)₀₋₄N(R^(o))C(O)OR^(o); —N(R^(o))N(R^(o))C(O)R^(o);—N(R^(o))N(R^(o))C(O)NR^(o) ₂; —N(R^(o))N(R^(o))C(O)OR^(o);—(CH₂)₀₋₄C(O)R^(o); —C(S)R^(o); —(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄C(O)SR^(o); —(CH₂)₀₋₄C(O)OSiR^(o) ₃; —(CH₂)₀₋₄OC(O)R;—OC(O)(CH₂)₀₋₄SR^(o), SC(S)SR^(o); —(CH₂)₀₋₄SC(O)R^(o);—(CH₂)₀₋₄C(O)NR^(o) ₂; —C(S)NR₂; —C(S)SR^(o); —SC(S)SR^(o),—(CH₂)₀₋₄OC(O)NR^(o) ₂; —C(O)N(OR^(o))R^(o); —C(O)C(O)RO;—C(O)CH₂C(O)R^(o); —C(NOR^(o))R^(o); —(CH₂)₀₋₄SSR^(o);—(CH₂)₀₋₄S(O)₂R^(o); —(CH₂)₀₋₄S(O)₂OR^(o); —(CH₂)₀₋₄OS(O)₂R^(o);—S(O)₂NR^(o) ₂; —(CH₂)₀₋₄S(O)R^(o); —N(R^(o))S(O)₂NR^(o) ₂;—N(R^(o))S(O)₂R^(o); —N(OR^(o))R^(o); —C(NH)NR^(o) ₂; —P(O)₂R^(o);—P(O)R^(o) ₂; —OP(O)R^(o) ₂; —OP(O)(OR^(o))₂; SiR^(o) ₃; —(C₁₋₄ straightor branched alkylene)O—N(R^(o))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(o))₂, wherein each R^(o) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(o), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(o) (or the ring formed by takingtwo independent occurrences of R^(o) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R., -(haloR.), —(CH₂)₀₋₂OH,—(CH₂)₀₋₂OR., —(CH₂)₀₋₂CH(OR.)₂; —O(haloR.), —CN, —N₃, —(CH₂)₀₋₂C(O)R.,—(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR., —(CH₂)₀₋₂SR., —(CH₂)₀₋₂SH,—(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR., —(CH₂)₀₋₂NR₁₂, —NO₂, —SiR₁₃, —OSiR.₃,—C(O)SR. —(C₁₋₄ straight or branched alkylene)C(O)OR., or —SSR. whereineach R. is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently selected from C₁₋₄aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(o) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen, —R.,-(haloR.), —OH, —OR., —O(haloR.), —CN, —C(O)OH, —C(O)OR., —NH₂, —NHR.,—NR₁₂, or —NO₂, wherein each R. is unsubstituted or where preceded by“halo” is substituted only with one or more halogens, and isindependently C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R., -(haloR.), —OH, —OR., —O(haloR.), —CN, —C(O)OH, —C(O)OR.,—NH₂, —NHR., —NR.₂, or —NO₂, wherein each R. is unsubstituted or wherepreceded by “halo” is substituted only with one or more halogens, and isindependently C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention. In someembodiments, the R¹ group of formula I-a and I-b comprises one or moredeuterium atoms.

As used herein, the term “irreversible” or “irreversible inhibitor”refers to an inhibitor (i.e. a compound) that is able to be covalentlybonded to a target protein kinase in a substantially non-reversiblemanner. That is, whereas a reversible inhibitor is able to bind to (butis generally unable to form a covalent bond) the target protein kinase,and therefore can become dissociated from the target protein kinase, anirreversible inhibitor will remain substantially bound to the targetprotein kinase once covalent bond formation has occurred. Irreversibleinhibitors usually display time dependency, whereby the degree ofinhibition increases with the time with which the inhibitor is incontact with the enzyme. Methods for identifying if a compound is actingas an irreversible inhibitor are known to one of ordinary skill in theart. Such methods include, but are not limited to, enzyme kineticanalysis of the inhibition profile of the compound with the proteinkinase target, the use of mass spectrometry of the protein drug targetmodified in the presence of the inhibitor compound, discontinuousexposure, also known as “washout,” experiments, and the use of labeling,such as radiolabelled inhibitor, to show covalent modification of theenzyme, as well as other methods known to one of skill in the art.

One of ordinary skill in the art will recognize that certain reactivefunctional groups can act as “warheads.” As used herein, the term“warhead” or “warhead group” refers to a functional group present on acompound of the present invention wherein that functional group iscapable of covalently binding to an amino acid residue (such ascysteine, lysine, histidine, or other residues capable of beingcovalently modified) present in the binding pocket of the targetprotein, thereby irreversibly inhibiting the protein. It will beappreciated that the -L-Y group, as defined and described herein,provides such warhead groups for covalently, and irreversibly,inhibiting the protein.

As used herein, the term “inhibitor” is defined as a compound that bindsto and/or inhibits the target protein kinase with measurable affinity.In certain embodiments, an inhibitor has an IC₅₀ and/or binding constantof less about 50 μM, less than about 1 μM, less than about 500 nM, lessthan about 100 nM, or less than about 10 nM.

The terms “measurable affinity” and “measurably inhibit,” as usedherein, means a measurable change in at least one of ErbB1, ErbB2,ErbB3, ErbB4, a TEC-kinase, and/or JAK3 activity between a samplecomprising a compound of the present invention, or composition thereof,and at least one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/orJAK3, and an equivalent sample comprising at least one of ErbB1, ErbB2,ErbB3, ErbB4, a TEC-kinase, and/or JAK3, in the absence of saidcompound, or composition thereof.

3. Description of Exemplary Compounds

According to one aspect, the present invention provides a compound offormula I,

or a pharmaceutically acceptable salt thereof, wherein:

-   Ring A is an optionally substituted group selected from phenyl, an    8-10 membered bicyclic partially unsaturated or aryl ring, a 5-6    membered monocyclic heteroaryl ring having 1-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, or an 8-10    membered bicyclic heteroaryl ring having 1-5 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   Ring B is phenyl, a 5-6 membered heteroaryl ring having 1-3    heteratoms independently selected from N, O or S, a 5-6 membered    saturated heterocyclic ring having 1-2 heteroatoms independently    selected from N, O or S, or an 8-10 membered bicyclic partially    unsaturated or aryl ring having 1-3 heteroatoms independently    selected from N, O or S;-   R¹ is -L-Y, wherein:    -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by cyclopropylene, —NR—, —N(R)C(O)—,        —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—,        —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with 1-4 R^(e)        groups; and    -   each R^(e) is independently selected from -Q-Z, oxo, NO₂,        halogen, CN, a suitable leaving group, or a C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—,            —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—,            or —SO₂N(R)—; and        -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with            oxo, halogen, NO₂, or CN;-   R^(y) is hydrogen, halogen, CN, lower alkyl, or lower haloalkyl;-   W is a bivalent C₁₋₃ alkylene chain wherein one methylene unit of W    is optionally replaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—,    —N(R²)SO₂—, —SO₂N(R²)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or    —SO₂—;-   R² is hydrogen or optionally substituted C₁₋₆ aliphatic, or:    -   R² and a substituent on Ring A are taken together with their        intervening atoms to form a 4-6 membered saturated ring, or:    -   R² and R^(y) are taken together with their intervening atoms to        form a 4-7 membered carbocyclic ring;-   m is 0-4;-   each R^(x) is independently selected from —R, halogen, —OR., —CN,    —NO₂, —SO₂R, —SO₂R, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂,    —NRSO₂R, or —N(R)₂; or:    -   R^(x) and R¹ are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group and 0-3 groups independently selected from        oxo, halogen, CN, or C₁₋₆ aliphatic; and-   each R group is independently hydrogen or an optionally substituted    group selected from C₁₋₆ aliphatic, phenyl, a 4-7 membered    heterocylic ring having 1-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl    ring having 1-4 heteroatoms independently selected from nitrogen,    oxygen, or sulfur.

In certain embodiments, the present invention provides a compound offormula II:

or a pharmaceutically acceptable salt thereof, wherein:

-   Ring A is an optionally substituted group selected from phenyl, an    8-10 membered bicyclic partially unsaturated or aryl ring, a 5-6    membered monocyclic heteroaryl ring having 1-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, or an 8-10    membered bicyclic heteroaryl ring having 1-5 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R¹ is -L-Y, wherein:    -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by cyclopropylene, —NR—, —N(R)C(O)—,        —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—,        —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with 1-4 R^(e)        groups; and    -   each R^(e) is independently selected from -Q-Z, oxo, NO₂,        halogen, CN, a suitable leaving group, or a C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—,            —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—,            or —SO₂N(R)—; and-   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,    halogen, NO₂, or CN;-   R^(y) is hydrogen, halogen, CN, lower alkyl, or lower haloalkyl;-   G is CH, or N;-   W is —NR²—, —S—, or —O—;-   R² is hydrogen or optionally substituted C₁₋₆ aliphatic, or:    -   R² and a substituent on Ring A are taken together with their        intervening atoms to form a 4-6 membered saturated ring;-   m is 0-4;-   each R^(x) is independently selected from —R, halogen, —OR, —CN,    —NO₂, —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂,    —NRSO₂R, or —N(R)₂; or:    -   R^(x) and R¹ are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group and 0-3 groups independently selected from        oxo, halogen, CN, or C₁₋₆ aliphatic; and-   each R group is independently hydrogen or an optionally substituted    group selected from C₁₋₆ aliphatic, phenyl, a 4-7 membered    heterocylic ring having 1-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl    ring having 1-4 heteroatoms independently selected from nitrogen,    oxygen, or sulfur.

According to one aspect, the present invention provides a compound offormula II-a or II-b:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,W, R¹, G, R^(y), R^(x) and m are as defined above for formula II and asdescribed herein.

In certain embodiments, the present invention provides a compound offormula II-b where in said compound is other thanN⁶-m-tolyl-N⁴-p-tolylpyrimidine-4,6-diamine.

In certain embodiments, the present invention provides a compound offormula II-a wherein said compound is other thanN⁴-(3-aminophenyl)-N⁶-(3-bromophenyl)pyrimidine-4,6-diamine,N-(3-(6-(3-(trifluoromethyl)phenylamino)pyrimidin-4-ylamino)phenyl)cyclopropane-carboxamide,N-(3-(6-(3-bromophenylamino)pyrimidin-4-ylamino)phenyl)propionamide,N⁴-(3-aminophenyl)-N⁶-m-tolylpyrimidine-4,6-diamine, orN⁴-(3-aminophenyl)-N⁶-methyl-N-6-phenyl-pyrimidine-4,6-diamine.

As defined generally above, the Ring A group of formulae I and II is anoptionally substituted group selected from phenyl, an 8-10 memberedbicyclic partially unsaturated or aryl ring, a 5-6 membered monocyclicheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, or sulfur. In certain embodiments, Ring A is an optionallysubstituted phenyl group. In some embodiments, Ring A is an optionallysubstituted naphthyl ring or a bicyclic 8-10 membered heteraryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In some embodiments, Ring A is an optionally substituteddiphenyl ether. In some embodiments, Ring A is an optionally substitutedphenyl benzyl ether. In other embodiments, Ring A is an optionallysubstituted pyridine methoxy phenyl group.

In certain embodiments, the Ring A group of formulae I and II issubstituted as defined herein. In some embodiments, Ring A issubstituted with one, two, or three groups independently selected fromhalogen, R^(o), or —(CH₂)₀₋₄OR^(o), or —O(CH₂)₀₋₄R^(o), wherein eachR^(o) is as defined herein. Exemplary substituents on Ring A include Br,I, Cl, methyl, —CF₃, —C≡CH, —OCH₂phenyl, —OCH₂(fluorophenyl), or—OCH₂pyridyl.

Exemplary Ring A groups of formulae I and II are set forth in Table 1.

TABLE 1 Exemplary Ring A Groups

i

ii

iii

iv

v

vi

vii

viii

ix

x

xi

xii

xiii

xiv

xv

xvi

xvii

xviii

xix

xx

xxi

xxii

xxiii

xxiv

xxv

xxvi

xxvii

xxviii

xxix

xxx

xxxi

xxxii

xxxiii

xxxiv

xxxv

xxxvi

xxxvii

xxxviii

xxxix

xl

xli

xlii

xliii

xliv

xlv

xlvi

xlvii

xlviii

xlix

l

li

lii

liii

liv

lv

lvi

As defined generally above, the Ring B group of formula I is phenyl, a5-6 membered heteroaryl ring having 1-3 heteratoms independentlyselected from N, O or S, a 5-6 membered saturated heterocyclic ringhaving 1-2 heteroatoms independently selected from N, O or S, or an 8-10membered bicyclic partially unsaturated or aryl ring having 1-3heteroatoms independently selected from N, O or S.

In some embodiments, the Ring B group of formula I is phenyl. In someembodiments, Ring B is a 6-membered heteroaryl ring having 1-3nitrogens. In some embodiments, Ring B is a 5-membered heteroaryl ringhaving 1 or 2 or 3 heteroatoms independently selected from N, O or S.

In some embodiments, the Ring B group of formula I is a 5-6 memberedsaturated heterocyclic ring having 1 nitrogen. In some embodiments, RingB is a 9-10 membered bicyclic partially saturated heteroaryl ring having1-3 nitrogens. In some embodiments, Ring B is a 9-10 membered bicyclicpartially saturated heteroaryl ring having 1 nitrogen.

Exemplary Ring B groups are set forth in Table 2.

TABLE 2 Ring B Groups

i

ii

iii

iv

v

vi

vii

viii

ix

x

xi

xii

xiii

xiv

In some embodiments, the m moiety of formula I, II, IIa, or IIb is 1, 2,3 or 4. In some embodiments, m is 1. In other embodiments, m is 0.

As defined generally above, each R^(x) group of formula I or II isindependently selected from —R, halogen, —OR, —CN, —NO₂, —SO₂R, —SOR,—C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)NR₂, —NRSO₂R, or —N(R)₂, or

-   -   R^(x) and R¹ are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group, wherein the warhead group is -Q-Z, and        said ring is further substituted with 0-3 groups independently        selected from oxo, halogen, CN, or C₁₋₆ aliphatic.

In some embodiments, each instance of R^(x) is independently selectedfrom —R, —OR or halogen. In certain embodiments, R^(x) is lower alkyl,lower alkoxy, or halogen. Exemplary R^(x) groups include methyl,methoxy, and chloro. In some embodiments, R^(x) is hydrogen.

In some embodiments, the G group of any of formula II, II-a, or II-b isCH. In other embodiments, the G group of any of formula II, II-a, orII-b is N.

As defined generally above, the W group of formula I is a bivalentC₁₋₃alkylene chain wherein one methylene unit of W is optionallyreplaced by —NR²—, —N(R²)C(O)—, —C(O)N(R²)—, —N(R²)SO₂—, —SO₂N(R²)—,—O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO— or —SO₂—.

In certain embodiments, the W group of formula I is —NH—, —S—, or —O—.In some embodiments, the W group of formula I is —CH₂O—, —CH₂S—, or—CH₂NH—. In some aspects, W is —OCH₂—, —SCH₂—, —NHCH₂—, or —CH₂CH₂—.

In some embodiments, the W group of formula I is —O— thus forming acompound of formula I-i:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R^(x), R^(y), and m are as defined above and described in classesand subclasses above and herein.

In some embodiments, W is —NR²— thus forming a compound of formula I-ii:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R^(x), R^(y), and m are as defined above and described inclasses and subclasses above and herein.

In some embodiments, W is —S— thus forming a compound of formula I-iii:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R^(x), R^(y), and m are as defined above and described in classesand subclasses above and herein.

In some embodiments, the W group of formula II is —O— thus forming acompound of formula II-i:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R^(x), R^(y), and m are as defined above and described in classesand subclasses above and herein.

In some embodiments, the W group of formula II is —NR²— thus forming acompound of formula II-ii:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R^(x), R^(y), and m are as defined above and described inclasses and subclasses above and herein.

In certain embodiments, R² is hydrogen. In some embodiments, R² ismethyl. In still other embodiments, R² is lower alkyl.

In some embodiments, the W group of formula II is —S— thus forming acompound of formula II-iii:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R^(x), R^(y), and m are as defined above and described in classesand subclasses above and herein.

In certain embodiments, the G group of formula II is CH, thus forming acompound of formula III:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,W, R¹, R^(x), R^(y), and m are as defined above and described in classesand subclasses above and herein.

In certain embodiments, the compound of formula III is of formulaIII-a-i, III-b-i, III-a-ii, III-b-ii, III-a-iii, or III-b-iii:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R^(x), R^(y), and m are as defined above and described inclasses and subclasses above and herein.

In certain embodiments, the G group of formula II is N, thus forming acompound of formula IV:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,W, R¹, R^(x), R^(y), and m are as defined above and described in classesand subclasses above and herein.

In certain embodiments, the compound of formula IV is of formula IV-a-i,IV-b-i, IV-a-ii, IV-b-ii, IV-a-iii, or IV-b-iii:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R^(x), R^(y), and m are as defined above and described inclasses and subclasses above and herein.

According to some aspects, R² and a substituent on Ring A are takentogether with their intervening atoms to form a 4-7 membered saturatedor partially unsaturated ring, thus forming a compound of formulaII-a-iv or II-b-iv:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R^(x), and m are as defined above and described in classes andsubclasses above and herein.

In certain embodiments, the present invention provides a compound offormula II-a-v or II-b-v wherein Ring A is phenyl and said compound isof formula II-a-v or II-b-v:

or a pharmaceutically acceptable salt thereof, wherein the Ring A phenylmoiety is optionally substituted and each of R¹, R^(x), R^(y), and m areas defined above and described in classes and subclasses above andherein.

In some embodiments, R² is hydrogen. In other embodiments, R² and R^(y)are taken together thereby forming a compound of formula II-a-vi orII-b-vi:

As defined generally above, the R¹ group of formulae I and II is -L-Y,wherein:

-   L is a covalent bond or a bivalent C₁₋₈ saturated or unsaturated,    straight or branched, hydrocarbon chain, wherein one, two, or three    methylene units of L are optionally and independently replaced by    cyclopropylene, —NR—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—,    —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—,    —N═N—, or —C(═N₂)—;-   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,    halogen, NO₂, or CN, or a 3-10 membered monocyclic or bicyclic,    saturated, partially unsaturated, or aryl ring having 0-3    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    and wherein said ring is substituted with 1-4 R^(e) groups; and-   each R^(e) is independently selected from -Q-Z, oxo, NO₂, halogen,    CN, a suitable leaving group, or a C₁₋₆ aliphatic optionally    substituted with oxo, halogen, NO₂, or CN, wherein:    -   Q is a covalent bond or a bivalent C₁₋₆ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one or two methylene units of Q are optionally and independently        replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or        —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, or —SO₂N(R)—; and    -   Z is hydrogen or C₁₋₁₆ aliphatic optionally substituted with        oxo, halogen, NO₂, or CN.

In certain embodiments, L is a covalent bond.

In certain embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain. In certain embodiments, L is—CH₂—.

In certain embodiments, L is a covalent bond, —CH₂—, —NH—, —CH₂NH—,—NHCH₂—, —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,—NHC(O)CH₂OC(O)—, or —SO₂NH—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and one or twoadditional methylene units of L are optionally and independentlyreplaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—,—SO₂—, —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,—N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and oneor two additional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, and one or two additionalmethylene units of L are optionally and independently replaced bycyclopropylene, —O—, —N(R)—, or —C(O)—.

As described above, in certain embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onedouble bond. One of ordinary skill in the art will recognize that such adouble bond may exist within the hydrocarbon chain backbone or may be“exo” to the backbone chain and thus forming an alkylidene group. By wayof example, such an L group having an alkylidene branched chain includes—CH₂C(═CH₂)CH₂—. Thus, in some embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onealkylidenyl double bond. Exemplary L groups include —NHC(O)C(═CH₂)CH₂—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—. In certain embodiments, Lis —C(O)CH═CH(CH₃)—, —C(O)CH═CHCH₂NH(CH₃)—, —C(O)CH═CH(CH₃)—,—C(O)CH═CH—, —CH₂C(O)CH═CH—, —CH₂C(O)CH═CH(CH₃)—, —CH₂CH₂C(O)CH═CH—,—CH₂CH₂C(O)CH═CHCH₂—, —CH₂CH₂C(O)CH═CHCH₂NH(CH₃)—, or—CH₂CH₂C(O)CH═CH(CH₃)—, or —CH(CH₃)OC(O)CH═CH—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —OC(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—,—SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and one or twoadditional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—. In some embodiments,L is —CH₂OC(O)CH═CHCH₂—, —CH₂—OC(O)CH═CH—, or —CH(CH═CH₂)OC(O)CH═CH—.

In certain embodiments, L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,—NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,—NRC(O)(C═N₂)C(O)—, —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—,—NRSO₂CH═CHCH₂—, —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,—CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-, whereineach R is independently hydrogen or optionally substituted C₁₋₆aliphatic.

In certain embodiments, L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,—NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,—NHC(O)(C═N₂)C(O)—, —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—,—NHSO₂CH═CHCH₂—, —NHC(O)CH═CHCH₂O—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,—CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one triple bond. In certainembodiments, L is a bivalent C₂₋₈ straight or branched, hydrocarbonchain wherein L has at least one triple bond and one or two additionalmethylene units of L are optionally and independently replaced by—NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—,or —C(O)—. In some embodiments, L has at least one triple bond and atleast one methylene unit of L is replaced by —N(R)—, —N(R)C(O)—, —C(O)—,—C(O)O—, or —OC(O)—, or —O—.

Exemplary L groups include —C≡C—, —C≡CCH₂N(isopropyl)-,—NHC(O)C≡CCH₂CH₂—, —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or—CH₂OC(═O)C≡C—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein one methylene unit of L is replaced bycyclopropylene and one or two additional methylene units of L areindependently replaced by —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, or—SO₂N(R)—. Exemplary L groups include —NHC(O)-cyclopropylene-SO₂— and—NHC(O)-cyclopropylene-.

As defined generally above, Y is hydrogen, C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN, or a 3-10 membered monocyclicor bicyclic, saturated, partially unsaturated, or aryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, andwherein said ring is substituted with at 1-4 R^(e) groups, each R^(e) isindependently selected from -Q-Z, oxo, NO₂, halogen, CN, a suitableleaving group, or C₁₋₆ aliphatic, wherein Q is a covalent bond or abivalent C₁₋₆ saturated or unsaturated, straight or branched,hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —N(R)—, —S—, —O—, —C(O)—,—OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, or—SO₂N(R)—; and, Z is hydrogen or C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN.

In certain embodiments, Y is hydrogen.

In certain embodiments, Y is C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN. In otherembodiments, Y is C₂₋₆ alkynyl optionally substituted with oxo, halogen,NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyl. In otherembodiments, Y is C₂₋₄alkynyl.

In other embodiments, Y is C₁₋₆ alkyl substituted with oxo, halogen,NO₂, or CN. Such Y groups include —CH₂F, —CH₂Cl, —CH₂CN, and —CH₂NO₂.

In certain embodiments, Y is a saturated 3-6 membered monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein Y is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein.

In some embodiments, Y is a saturated 3-4 membered heterocyclic ringhaving 1 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-2 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Exemplary such rings are epoxide and oxetanerings, wherein each ring is substituted with 1-2 R^(e) groups, whereineach R^(e) is as defined above and described herein.

In other embodiments, Y is a saturated 5-6 membered heterocyclic ringhaving 1-2 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-4 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Such rings include piperidine andpyrrolidine, wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R, Q, Z, and R^(e) is as defined above and describedherein.

In some embodiments, Y is a saturated 3-6 membered carbocyclic ring,wherein said ring is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein. In certain embodiments,Y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein. In certain embodiments, Y is

wherein R^(e) is as defined above and described herein. In certainembodiments, Y is cyclopropyl optionally substituted with halogen, CN orNO₂.

In certain embodiments, Y is a partially unsaturated 3-6 memberedmonocyclic ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein.

In some embodiments, Y is a partially unsaturated 3-6 memberedcarbocyclic ring, wherein said ring is substituted with 1-4 R^(e)groups, wherein each R^(e) is as defined above and described herein. Insome embodiments, Y is cyclopropenyl, cyclobutenyl, cyclopentenyl, orcyclohexenyl wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R^(e) is as defined above and described herein.

In certain embodiments, Y is a partially unsaturated 4-6 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein. In certain embodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is a 6-membered aromatic ring having 0-2nitrogens wherein said ring is substituted with 1-4 R^(e) groups,wherein each R^(e) group is as defined above and described herein. Incertain embodiments, Y is phenyl, pyridyl, or pyrimidinyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein.

In some embodiments, Y is selected from:

wherein each R^(e) is as defined above and described herein.

In other embodiments, Y is a 5-membered heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein said ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In someembodiments, Y is a 5 membered partially unsaturated or aryl ring having1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein said ring is substituted with 1-4R^(e) groups, whereineach R^(e) group is as defined above and described herein. Exemplarysuch rings are isoxazolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,pyrrolyl, furanyl, thienyl, triazole, thiadiazole, and oxadiazole,wherein each ring is substituted with 1-3R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In certainembodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is an 8-10 membered bicyclic, saturated,partially unsaturated, or aryl ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein said ring issubstituted with 1-4 R^(e) groups, wherein R^(e) is as defined above anddescribed herein. According to another aspect, Y is a 9-10 memberedbicyclic, partially unsaturated, or aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is substituted with 1-4 R^(e) groups, wherein R^(e) is as definedabove and described herein. Exemplary such bicyclic rings include2,3-dihydrobenzo[d]isothiazole, wherein said ring is substituted with1-4 R^(e) groups, wherein R^(e) is as defined above and describedherein.

As defined generally above, each R^(e) group is independently selectedfrom -Q-Z, oxo, NO₂, halogen, CN, a suitable leaving group, or C₁₋₆aliphatic optionally substituted with oxo, halogen, NO₂, or CN, whereinQ is a covalent bond or a bivalent C₁₋₆ saturated or unsaturated,straight or branched, hydrocarbon chain, wherein one or two methyleneunits of Q are optionally and independently replaced by —N(R)—, —S—,—O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—,—N(R)SO₂—, or —SO₂N(R)—; and Z is hydrogen or C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN.

In certain embodiments, R^(e) is C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN. In other embodiments, R^(e) is oxo, NO₂,halogen, or CN.

In some embodiments, R^(e) is -Q-Z, wherein Q is a covalent bond and Zis hydrogen (i.e., R^(e) is hydrogen). In other embodiments, R^(e) is-Q-Z, wherein Q is a bivalent C₁₋₆ saturated or unsaturated, straight orbranched, hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —NR—, —NRC(O)—, —C(O)NR—, —S—,—O—, —C(O)—, —SO—, or —SO₂—. In other embodiments, Q is a bivalent C₂₋₆straight or branched, hydrocarbon chain having at least one double bond,wherein one or two methylene units of Q are optionally and independentlyreplaced by —NR—, —NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—.In certain embodiments, the Z moiety of the R^(e) group is hydrogen. Insome embodiments, -Q-Z is —NHC(O)CH═CH₂ or —C(O)CH═CH₂.

In certain embodiments, each R^(e) is independently selected from oxo,NO₂, CN, fluoro, chloro, —NHC(O)CH═CH₂, —C(O)CH═CH₂, —CH₂CH═CH₂, —C≡CH,—C(O)OCH₂Cl, —C(O)OCH₂F, —C(O)OCH₂CN, —C(O)CH₂Cl, —C(O)CH₂F, —C(O)CH₂CN,or —CH₂C(O)CH₃.

In certain embodiments, R^(e) is a suitable leaving group, ie a groupthat is subject to nucleophilic displacement. A “suitable leaving” is achemical group that is readily displaced by a desired incoming chemicalmoiety such as the thiol moiety of a cysteine of interest. Suitableleaving groups are well known in the art, e.g., see, “Advanced OrganicChemistry,” Jerry March, 5^(th) Ed., pp. 351-357, John Wiley and Sons,N.Y. Such leaving groups include, but are not limited to, halogen,alkoxy, sulphonyloxy, optionally substituted alkylsulphonyloxy,optionally substituted alkenylsulfonyloxy, optionally substitutedarylsulfonyloxy, acyl, and diazonium moieties. Examples of suitableleaving groups include chloro, iodo, bromo, fluoro, acetoxy,smethanesulfonyloxy (mesyloxy), tosyloxy, triflyloxy,nitro-phenylsulfonyloxy (nosyloxy), and bromo-phenylsulfonyloxy(brosyloxy).

In certain embodiments, the following embodiments and combinations of-L-Y apply:

-   -   (a) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (b) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,        —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—,        and one or two additional methylene units of L are optionally        and independently replaced by cyclopropylene, —O—, —N(R)—, or        —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (c) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, and one or two        additional methylene units of L are optionally and independently        replaced by cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y is        hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (d) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (e) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —OC(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (f) L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,        —NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—,        —NRSO₂CH═CHCH₂—, —NRC(O)(C═N₂)—, —NRC(O)(C═N₂)C(O)—,        —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,        —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,        —CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-;        wherein R is H or optionally substituted C₁₋₆ aliphatic; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (g) L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,        —NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—,        —NHSO₂CH═CHCH₂—, —NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—,        —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,        —NHC(O)CH═CHCH₂O—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,        —CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-;        and Y is hydrogen or C₁₋₆ aliphatic optionally substituted with        oxo, halogen, NO₂, or CN; or    -   (h) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one alkylidenyl double bond and at least        one methylene unit of L is replaced by —C(O)—, —NRC(O)—,        —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or        —C(O)O—, and one or two additional methylene units of L are        optionally and independently replaced by cyclopropylene, —O—,        —N(R)—, or —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (i) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one triple bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, or —C(O)O—, and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (j) L is —C≡C—, —C≡CCH₂N(isopropyl)-, —NHC(O)C≡CCH₂CH₂—,        —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or        —CH₂C(═O)C≡C—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (k) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein one methylene unit of L is replaced by cyclopropylene        and one or two additional methylene units of L are independently        replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,        —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—; and Y is hydrogen or C₁₋₆        aliphatic optionally substituted with oxo, halogen, NO₂, or CN;        or    -   (l) L is a covalent bond and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (m) L is —C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (n) L is —N(R)C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (o) L is a bivalent C₁₋₈ saturated or unsaturated, straight or        branched, hydrocarbon chain; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (p) L is a covalent bond, —CH₂—, —NH—, —C(O)—, —CH₂NH—, —NHCH₂—,        —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,        —NHC(O)CH₂OC(O)—, or —SO₂NH—; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein.

In certain embodiments, the Y group of formula I is selected from thoseset forth in Table 3, below, wherein each wavy line indicates the pointof attachment to the rest of the molecule.

TABLE 3 Exemplary Y groups:

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

vv

ww

xx

yy

zz

aaa

bbb

ccc

ddd

eee

fff

ggg

hhh

iii

jjj

kkk

lll

mmm

nnn

ooo

ppp

qqq

rrr

sss

ttt

uuu

vvv

qqq

www

xxx

yyy

zzz

aaaa

bbbb

cccc

dddd

eeee

ffff

gggg

hhhh

iiii

jjjj

kkkk

llll

mmmm

nnnn

oooo

pppp

qqqq

rrrr

ssss

tttt

uuuu

vvvv

wwww

xxxx

yyyy

zzzz

aaaaa

bbbbb

cccccwherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

In certain embodiments, R¹ is —C≡CH, —C≡CCH₂NH(isopropyl),—NHC(O)C≡CCH₂CH₃, —CH₂—C≡CCH₃, —C≡CCH₂OH, —CH₂C(O)C≡CH, —C(O)C≡CH, or—CH₂OC(═O)C≡CH. In some embodiments, R¹ is selected from —NHC(O)CH═CH₂,—NHC(O)CH═CHCH₂N(CH₃)₂, Or —CH₂NHC(O)CH═CH₂.

In certain embodiments, R¹ is selected from those set forth in Table 4,below, wherein each wavy line indicates the point of attachment to therest of the molecule.

TABLE 4 Exemplary R¹ Groups

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

vv

ww

xx

yy

zz

aaa

bbb

ccc

ddd

eee

fff

ggg

hhh

iii

jjj

kkk

lll

mmm

nnn

ooo

ppp

qqq

rrr

sss

ttt

uuu

vvv

www

xxx

yyy

zzz

aaaa

bbbb

cccc

dddd

eeee

ffff

gggg

hhhh

iiii

jjjj

kkkk

llll

mmmm

nnnn

oooo

pppp

qqqq

rrrr

ssss

tttt

uuuu

vvvv

wwww

xxxx

yyyy

zzzz

aaaaa

bbbbb

ccccc

ddddd

eeeee

fffff

ggggg

hhhhh

iiiii

jjjjj

kkkkk

lllll

mmmmm

nnnnn

ooooo

ppppp

qqqqq

rrrrr

sssss

ttttt

uuuuu

vvvvv

wwwww

xxxxx

yyyyy

zzzzz

aaaaaa

bbbbbb

cccccc

dddddd

eeeeee

ffffff

gggggg

hhhhhh

iiiiii

jjjjjj

kkkkkk

llllll

mmmmmm

nnnnnn

oooooo

pppppp

qqqqqq

rrrrrr

ssssss

tttttt

uuuuuu

vvvvvv

wwwwww or

xxxxxxwherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

As defined generally above, R¹ is a warhead group, or, when R¹ and R^(x)form a ring, then -Q-Z is a warhead group. Without wishing to be boundby any particular theory, it is believed that such R¹ groups, i.e.warhead groups, are particularly suitable for covalently binding to akey cysteine residue in the binding domain of certain protein kinases.Protein kinases having a cysteine residue in the binding domain areknown to one of ordinary skill in the art and include ErbB1, ErbB2, andErbB4, or a mutant thereof. In certain embodiments, compounds of thepresent invention have a warhead group characterized in that inventivecompounds target one or more of the following cysteine residues:

SEQ ID 7: ERBB1 ITQLMPFG C LLDYVREH SEQ ID 8: ERBB2 VTQLMPYG C LLDHVRENSEQ ID 9: ERBB4 VTQLMPHG C LLEYVHEH

Thus, in some embodiments, R¹ is characterized in that the -L-Y moietyis capable of covalently binding to a cysteine residue therebyirreversibly inhibiting the enzyme. In certain embodiments, the cysteineresidue is Cys797 of ErbB1, Cys805 of ErbB2 and Cys803 of ErbB4, or amutant thereof, where the provided residue numbering is in accordancewith Uniprot (code POO533 for ErbB1; code PO4626 for ErbB2, and Q15303for ErbB4). It will be understood that the Cys of ErbB1 (EGFR) isvariably called 773 or 797 depending on whether the parent sequencecontains the signal peptide or not. Thus, in accordance with the presentinvention, the relevant cysteine residue of ErbB1 may be described asCys 773 or Cys 797 and these terms are used interchangeably.

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding. Such R¹ groups include, but are not limited to, those describedherein and depicted in Table 4, infra. One of ordinary skill in the artwill recognize that ErbB3 has no corresponding residue and, asrecognized in the relevant art, is not catalytically active.

As depicted in Formula I supra, the R¹ warhead group can be in anortho-, meta-, or para-position. In certain embodiments, the R¹ warheadgroup is in a meta-position of the phenyl ring relative to the rest ofthe molecule. Without wishing to be bound by any particular theory, itis believed that when R¹ is in such a meta-position, the warhead groupis better positioned for covalent modification of the cysteine residuethus effecting irreversible inhibition of the enzyme. Indeed, it hasbeen surprisingly found that a compound having a warhead group at ameta-position (compound I-1) irreversibly binds to ErbB1 whereas acompound having a warhead group at a para-position (compound I-93)reversibly binds to ErbB1. These compounds have the followingstructures:

This phenomenon was determined by performing a washout experiment usingthe protocol described in detail in Example 42, infra. The results ofthis experiment are depicted in FIG. 2 where it is shown that compoundI-1 maintains enzyme inhibition after “washout” whereas compound I-93was washed away in the experiment thereby resulting in reactivatedenzyme activity.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of TEC, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 449.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of BTK, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 481.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of ITK, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 442.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of BMX, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 496.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of JAK3, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 909.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of TXK, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 350.

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding. Such R¹ groups include, but are not limited to, those describedherein and depicted in Table 3, infra.

Exemplary compounds of formula I are set forth in Table 5 below.

TABLE 5 Exemplary Compounds of Formula I

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-40

I-41

I-42

I-43

I-44

I-45

I-46

I-47

I-48

I-49

I-50

I-51

I-52

I-53

I-54

I-55

I-56

I-57

I-58

I-59

I-60

I-61

I-62

I-63

I-64

I-65

I-66

I-67

I-68

I-69

I-70

I-71

I-72

I-73

I-74

I-75

I-76

I-77

I-78

I-79

I-80

I-81

I-82

I-83

I-84

I-85

I-86

I-87

I-88

I-89

I-90

I-91

I-92

I-93

I-94

I-95

I-96

I-97

I-98

I-99

I-100

I-101

I-102

I-103

I-104

I-105

I-106

I-107

I-108

I-109

I-110

I-111

I-112

I-113

I-114

I-115

I-116

I-117

I-118

I-119

I-120

I-121

In certain embodiments, the present invention provides any compounddepicted in Table 5, above, or a pharmaceutically acceptable saltthereof.

In certain embodiments, the present invention provides a compoundselected from:

or a pharmaceutically acceptable salt thereof.

As described herein, compounds of the present invention are irreversibleinhibitors of at least one of ErbB1, ErbB2, ErbB3 and ErbB4, or a mutantthereof. In some embodiments, provided compounds are irreversibleinhibitors of a TEC-kinase (e.g. BTK) and JAK3. One of ordinary skill inthe art will recognize that certain compounds of the present inventionare reversible inhibitors. In certain embodiments, such compounds areuseful as assay comparator compounds. In other embodiments, suchreversible compounds are useful as inhibitors of ErbB1, ErbB2, ErbB3,ErbB4, a TEC-kinase, and/or JAK3, or a mutant thereof, and thereforeuseful for treating one or disorders as described herein. Exemplaryreversible compounds of the present invention are set forth in Table 6,below.

TABLE 6 Reversible Inhibitors

I^(R)-1

I^(R)-2

I^(R)-3

I^(R)-4

I^(R)-5

I^(R)-6

I^(R)-7

I^(R)-8

I^(R)-9

I^(R)-10

I^(R)-11

I^(R)-12

I^(R)-13

I^(R)-14

I^(R)-15

I^(R)-16

I^(R)-17

I^(R)-18or a pharmaceutically acceptable salt thereof.

4. Uses, Formulation and Administration Pharmaceutically AcceptableCompositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit a protein kinase,particularly at least one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase,and/or JAK3, or a mutant thereof, in a biological sample or in apatient. In certain embodiments, the amount of compound in compositionsof this invention is such that is effective to measurably inhibit atleast one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or amutant thereof, in a biological sample or in a patient. In certainembodiments, a composition of this invention is formulated foradministration to a patient in need of such composition. In someembodiments, a composition of this invention is formulated for oraladministration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof at least one of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/orJAK3, or a mutant thereof.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. Preferably, provided compositions should be formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of protein kinase activity of one or more enzymes.

Drug resistance is emerging as a significant challenge for targetedtherapies. For example, drug resistance has been reported for Gleevec®and Iressa®, as well as several other kinase inhibitors in development.In addition, drug resistance has been reported for the cKit and PDGFRreceptors. It has been reported that irreversible inhibitors may beeffective against drug resistant forms of protein kinases (Kwak, E. L.,R. Sordella, et al. (2005). “Irreversible inhibitors of the EGF receptormay circumvent acquired resistance to gefitinib.” PNAS 102(21):7665-7670.) Without wishing to be bound by any particular theory, it isbelieved that compounds of the present invention may be effectiveinhibitors of drug resistant forms of protein kinases.

As used herein, the term “clinical drug resistance” refers to the lossof susceptibility of a drug target to drug treatment as a consequence ofmutations in the drug target.

As used herein, the term “resistance” refers to changes in the wild-typenucleic acid sequence coding a target protein, and/or the proteinsequence of the target, which change, decrease or abolish the inhibitoryeffect of the inhibitor on the target protein.

Examples of kinases that are inhibited by the compounds and compositionsdescribed herein and against which the methods described herein areuseful include ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, ora mutant thereof.

The activity of a compound utilized in this invention as an inhibitor ofErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or a mutantthereof, may be assayed in vitro, in vivo or in a cell line. In vitroassays include assays that determine inhibition of either thephosphorylation activity and/or the subsequent functional consequences,or ATPase activity of activated ErbB1, ErbB2, ErbB3, ErbB4, aTEC-kinase, and/or JAK3, or a mutant thereof. Alternate in vitro assaysquantitate the ability of the inhibitor to bind to ErbB1, ErbB2, ErbB3,ErbB4, a TEC-kinase, and/or JAK3. Inhibitor binding may be measured byradiolabeling the inhibitor prior to binding, isolating theinhibitor/ErbB1, inhibitor/ErbB2, inhibitor/ErbB3, inhibitor/ErbB4,inhibitor/TEC-kinase, or inhibitor/JAK3 complex and determining theamount of radiolabel bound. Alternatively, inhibitor binding may bedetermined by running a competition experiment where new inhibitors areincubated with ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3bound to known radioligands. Detailed conditions for assaying a compoundutilized in this invention as an inhibitor of ErbB1, ErbB2, ErbB3,ErbB4, a TEC-kinase, and/or JAK3, or a mutant thereof, are set forth inthe Examples below.

Protein tyrosine kinases are a class of enzymes that catalyze thetransfer of a phosphate group from ATP or GTP to a tyrosine residuelocated on a protein substrate. Receptor tyrosine kinases act totransmit signals from the outside of a cell to the inside by activatingsecondary messaging effectors via a phosphorylation event. A variety ofcellular processes are promoted by these signals, includingproliferation, carbohydrate utilization, protein synthesis,angiogenesis, cell growth, and cell survival.

ErbB receptors, a major family of receptor tyrosine kinases, arecomposed of an extracellular ligand binding domain, a singletransmembrane domain, and an intracellular domain with tyrosine kinaseactivity. The ErbB family comprises ErbB1 (commonly known as EGFR),ErbB2 (commonly known as HER2 or neu), ErbB3 (commonly known as HER3),and ErbB4 (commonly known as HER4). More than 10 ligands (including EGF,TGFα, AR, BTC, EPR, HB-EGF, NRG-1, NRG-2, NRG-3, NRG-4) have beenidentified for the various receptor family members. Upon ligand bindingthe extracellular domain undergoes conformational change, allowing theformation of homodimers or heterodimers with other members of the ErbBfamily. Dimerization induces tyrosine phosphorylation of specificresidues in the intracellular domain that serve as docking sites foradaptor proteins and downstream effectors. In some contexts, activationof phosphatidyl-inositol 3-kinase (PI3K) and mitogen-activated proteinkinase pathways occur, leading to cell proliferation and survival (Lin,N. U.; Winer, E. P., Breast Cancer Res 6: 204-210, 2004).

Interaction between family members is necessitated by deficiencies inErbB2, which has no known ligand, and ErbB3, which is kinase dead. EGFR,ErbB3, and ErbB4 bind ligand to induce ErbB receptor homodimerization orheterodimerization, whereas ErbB2 functions as the preferreddimerization partner. The composition of the pairwise combinations isimportant for signal diversification, as dimer identity determines whichdownstream pathways are activated. Representative downstream geneproducts in the ErbB signal transduction pathway include Shc, Grb2,SOS1, Ras, Raf1, Mek, ERK1, ERK2, ERα, Akt, mTOR, FKHR, p27, Cyclin D1,FasL, GSK-3, Bad, and STAT3.

There is strong precedent for involvement of the EGFR and other membersof the ErbB family in human cancer because over 60% of all solid tumorsoverexpress at least one of these proteins or their ligands.Constitutively active, tumorigenic EGFR vIII, a mutant possessing atruncated extracellular domain, has been reported to be present in up to78% of breast carcinomas and has also been found in glioblastomas.Overexpression of EGFR is commonly found in breast, lung, head and neck,bladder tumors, while ErbB2 expression is frequently elevated in humantumors of epithelial origin. Activating mutations in the tyrosine kinasedomain have been identified in patients with non-small cell lung cancer(Lin, N. U.; Winer, E. P., Breast Cancer Res 6: 204-210, 2004). ErbB1and/or ErbB2 amplification has also been implicated in squamous cellcarcinomas, salivary gland carcinomas, ovarian carcinomas, andpancreatic cancers (Cooper, G. C. Oncogenes. 2^(nd) ed. Sudbury: Jonesand Barlett, 1995; Zhang, Y., et al., Cancer Res 66: 1025-32, 2006).Overexpression of ErbB2 has potent transforming activity, likely due toits ability to cooperate with other ErbB receptors (Sherman, L., et al.,Oncogene 18: 6692-99, 1999). In fact, some human cancers thatoverexpress both EGFR and ErbB2 have a poorer prognosis than cancersthat overexpress either receptor alone.

The ErbB signaling network is often a key component in the pathogenesisof breast cancer. Amplification of ErbB2 is associated with anaggressive tumor phenotype that is characterized by relatively rapidtumor growth, metastatic spread to visceral sites, and drug resistance.ErbB2 has been shown to be amplified in 20% of axillary node-negative(“ANN”) breast cancer cases, and this amplification has been identifiedas an independent prognostic factor for risk of recurrence in ANN breastcancer. (Andrulis, I. L., et al., J Clin Oncol 16: 1340-9, 1998).

Targeted blockade of ErbB signaling with trastuzumab (Herceptin), amonoclonal antibody directed at ErbB2, has been shown to improvesurvival in women with ErbB2-positive, advanced breast cancer. Othermonoclonal antibodies directed against ErbB receptors include cetuximab(Erbitux) and panitumumab (Vectibix).

Several small molecule tyrosine kinase inhibitors (TKIs) have been foundto act selectively upon ErbB family members. Notable examples includegefitinib (Iressa) and erlotinib (Tarceva), both of which target theEGFR. These small molecules compete with ATP for binding to the kinasedomain of the receptor. Compared to monoclonal antibodies, TKIs haveseveral advantages in that they are orally bioavailable, well-tolerated,and appear to be active against truncated forms of ErbB2 and EGFRreceptors (e.g., EGFR vIII) in vitro. In addition, the small size ofsmall molecule TKIs may allow them to penetrate sanctuary sites such asthe central nervous system. Finally, the homology between kinase domainsof ErbB receptors allows for development of TKIs that target more thanone member of the ErbB family simultaneously, the advantages of whichare described herein.

Although certain malignancies have been linked to the overexpression ofindividual receptors, efficient signal transduction relies on thecoexpression of ErbB receptor family members. This cooperation of ErbBreceptor family members in signal transduction and malignanttransformation may limit the success of agents that target individualreceptors in the treatment of cancer; a potential mechanism ofresistance to agents targeting a single ErbB receptor is upregulation ofother members of the receptor family (Britten, C. D., Mol Cancer Ther 3:1335-42, 2004).

Agents that target two or more ErbB receptors are called pan-ErbBregulators. ERRP is a pan-ErbB negative regulator that is expressed inmost benign pancreatic ductal epithelium and islet cells. Tumors havebeen found to experience a progressive loss in ERRP expression. Pan-ErbBregulators may be more successful in treating tumors than compounds thatonly target one ErbB receptor. Erbitux and Herceptin show success in alimited patient base (tumors having increased expression of EGFR orErbB2), which could be partly due to lack of pan-ErbB activity.

In both in vitro and in vivo models, strategies that employ a dual ErbBapproach seem to have greater antitumor activity than agents targeting asingle ErbB receptor. Thus, agents that target multiple members of theErbB family are likely to provide therapeutic benefit to a broaderpatient population (Zhang, Y., et al., Cancer Res 66: 1025-32, 2006). Incertain embodiments, provided compounds inhibit one or more of ErbB1,ErbB2, ErbB3, and ErbB4. In some embodiments, provided compounds inhibittwo or more of ErbB1, ErbB2, ErbB3, and ErbB4, or a mutant thereof, andare therefore pan-ErbB inhibitors.

Clearly, there is growing evidence to support the concurrent inhibitionof two or more ErbB (i.e., pan-ErbB) receptors in cancer therapy.Possible pan-ErbB approaches with small molecules include usingcombinations of agents that target individual ErbB receptors, usingsingle agents that target multiple ErbB receptors, or using agents thatinterfere with ErbB receptor interactions (e.g., dimerization).Additional strategies include therapies utilizing a small molecule incombination with antibodies, or chemoprevention therapies (Lin, N. U.;Winer, E. P., Breast Cancer Res 6: 204-210, 2004).

An example of small molecule pan-ErbB inhibition is CI-1033, anirreversible pan-ErbB inhibitor that covalently binds to the ATP bindingsite of the intracellular kinase domain. Another irreversible pan-ErbBreceptor tyrosine kinase inhibitor is HKI-272, which inhibits the growthof tumor cells that express ErbB-1 (EGFR) and ErbB-2 (HER-2) in cultureand xenografts, and has antitumor activity in HER-2-positive breastcancer (Andrulis, I. L., et al., J Clin Oncol 16: 1340-9, 1998).Irreversible inhibitors have demonstrated superior antitumor activity incomparison with reversible inhibitors.

Neurofibromatosis type I (NF1) is a dominantly inherited human diseaseaffecting one in 2500-3500 individuals. Several organ systems areaffected, including bones, skin, iris, and the central nervous system,as manifested in learning disabilities and gliomas. A hallmark of NF1 isthe development of benign tumors of the peripheral nervous system(neurofibromas), which vary greatly in both number and size amongpatients. Neurofibromas are heterogeneous tumors composed of Schwanncells, neurons, fibroblasts and other cells, with Schwann cells beingthe major (60-80%) cell type.

Abberant expression of the EGFR is associated with tumor development inNF1 and in animal models of NF1, suggesting a role in pathogenesis andrepresenting a novel potential therapeutic target. EGFR expressionaffects the growth of tumor cell lines derived from NF1 patients underconditions where EGF is not the primary factor driving growth of thecells. These data suggest that EGFR may play an important role in NF1tumorigenesis and Schwann cell transformation (DeClue, J. E., et al., JClin Invest 105: 1233-41, 2000).

Patients with NF1 develop aggressive Schwann cell neoplasms known asmalignant peripheral nerve sheath tumors (MPNSTs). Schwann cells are themajor supportive cell population in the peripheral nervous system.Neoplastic Schwann cells within these neoplasms variably express theErbB tyrosine kinases mediating NRG-1 responses (ErbB2, ErbB3, ErbB4).Neuregulin-1 (NRG-1) proteins promote the differentiation, survival,and/or proliferation of many cell types in the developing nervoussystem, and overexpression of NRG-1 in myelinating Schwann cells inducesthe formation of malignant peripheral nerve sheath tumors (MPNSTs)(Fallon, K. B., et al., J Neuro Oncol 66: 273-84, 2004).

Deregulation of Schwann cell growth is a primary defect driving thedevelopment of both benign neurofibromas and MPNST in neurofibromatosistype I (NF1) patients. Growth of MPNSTs and transformed mouse Schwanncells in vitro is highly EGF-dependent and can be blocked by EGFRinhibitors under conditions where EGF is the primary growth factor. Somehuman MPNST cell lines have been found to demonstrate constitutive ErbBphosphorylation. While treatment with ErbB inhibitors abolishes ErbBphosphorylation and reduces DNA synthesis in these lines, effectivechemotherapeutic regimens for MPNST remain elusive (Stonecypher, M. S.,et al., Oncogene 24: 5589-5605, 2005).

Schwannomas are peripheral nerve tumors comprised almost entirely ofSchwann-like cells, and typically have mutations in theneurofibromatosis type II (NF2) tumor suppressor gene. Ninety percent ofNF2 patients develop bilateral vestibular schwannomas and/or spinalschwannomas. Enlarging schwannomas can compress adjacent structures,resulting in deafness and other neurologic problems. Surgical removal ofthese tumors is difficult, often resulting in increased patientmorbidity.

Both normal human Schwann cells and schwannoma cells express neuregulinreceptors (i.e., ErbB receptors), and schwannoma cells proliferate inresponse to neuregulin. It is possible that aberrant neuregulinproduction or response contributes to aberrant schwannoma cellproliferation (Pelton, P. D., et al., Oncogene 17: 2195-2209, 1998).

The NF2 tumor suppressor, Merlin, is a membrane/cytoskeleton-associatedprotein implicated in the regulation of tyrosine kinase activity.Genetic interactions between a Merlin mutation and EGFR pathwaymutations have been documented in Drosophila (LaJeunesse, D. R., et al.,Genetics 158: 667-79, 2001). Other evidence suggests Merlin can inhibitEGFR internalization and signaling upon cell-cell contact by restrainingthe EGFR into a membrane compartment from which it can neither signalnor be internalized (McClatchey, A. I., et al., Genes and Development19: 2265-77, 2005; Curto, M. C., et al., J Cell Biol 177: 893-903,2007).

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

Provided compounds are inhibitors of one of more of ErbB1, ErbB2, ErbB3,and ErbB4 and are therefore useful for treating one or more disordersassociated with activity of one of more of ErbB1, ErbB2, ErbB3, andErbB4. Thus, in certain embodiments, the present invention provides amethod for treating an ErbB1-mediated, an ErbB2-mediated, anErbB3-mediated, and/or ErbB4-mediated disorder comprising the step ofadministering to a patient in need thereof a compound of the presentinvention, or pharmaceutically acceptable composition thereof.

As used herein, the terms “ErbB-mediated”, “ErbB2-mediated,”“ErbB3-mediated,” and/or “ErbB4-mediated” disorders or conditions asused herein means any disease or other deleterious condition in whichone or more of ErbB1, ErbB2, ErbB3, and/or ErbB4, or a mutant thereof,are known to play a role. Accordingly, another embodiment of the presentinvention relates to treating or lessening the severity of one or morediseases in which one or more of ErbB1, ErbB2, ErbB3, and/or ErbB4, or amutant thereof, are known to play a role. Specifically, the presentinvention relates to a method of treating or lessening the severity of adisease or condition selected from a proliferative disorder, whereinsaid method comprises administering to a patient in need thereof acompound or composition according to the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more disorders selectedfrom a cancer. In some embodiments, the cancer is associated with asolid tumor. In certain embodiments, the cancer is breast cancer,glioblastoma, lung cancer, cancer of the head and neck, colorectalcancer, bladder cancer, or non-small cell lung cancer. In someembodiments, the present invention provides a method for treating orlessening the severity of one or more disorders selected from squamouscell carcinoma, salivary gland carcinoma, ovarian carcinoma, orpancreatic cancer.

In certain embodiments, the present invention provides a method fortreating or lessening the severity of neurofibromatosis type I (NF1),neurofibromatosis type II (NF2) Schwann cell neoplasms (e.g. MPNST's),or Schwannomas.

The TEC family of non-receptor tyrosine kinases, referred to herein as“TEC-kinases,” plays a central role in signaling throughantigen-receptors such as the TCR, BCR and Fee receptors (reviewed inMiller A, et al. Current Opinion in Immunology 14; 331-340 (2002).TEC-kinases are essential for T cell activation. Three members of thefamily, Itk, Rlk and, are activated downstream of antigen receptorengagement in T cells and transmit signals to downstream effectors,including PLC-g. Combined deletion of Itk and Rlk in mice leads to aprofound inhibition of TCR responses including proliferation, cytokineproduction and immune responses to an intracellular parasite (Toxoplasmagondii) (Schaeffer et al, Science 284; 638-641 (1999)). Intracellularsignalling following TCR engagement is effected in ITK/RLK deficient Tcells; inositol triphosphate production, calcium mobilization and MAPkinase activation are all reduced. Tec-kinases are also essential for Bcell development and activation.

TEC-kinases include five family members, which are expressed primarilyin hematopoietic cells: TEC, BTK, ITK (also known as TSK and EMT), RLK(also known as TXK), and BMX (also known as ETK). Additional relatedTEC-kinases have been found in Drosophila melanogaster, zebrafish (Daniorerió), skate (Raja eglanteria), and sea urchin (Anthocidariscrassispina).

Provided compounds are inhibitors of one of more TEC-kinases and aretherefore useful for treating one or more disorders associated withactivity of one or more TEC-kinases. Thus, in certain embodiments, thepresent invention provides a method for treating a TEC-mediated disordercomprising the step of administering to a patient in need thereof acompound of the present invention, or pharmaceutically acceptablecomposition thereof.

The term “TEC-mediated condition”, as used herein means any disease orother deleterious condition in which TEC-kinases are known to play arole. Such conditions include those described herein and in Melcher, Met al., “The Role of TEC Family Kinases in Inflammatory Processes”,Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, Vol. 6,No. 1, pp. 61-69 (February 2007). Accordingly, another embodiment of thepresent invention relates to treating or lessening the severity of oneor more diseases in which TEC-kinases are known to play a role.Specifically, the present invention relates to a method of treating orlessening the severity of a disease or condition selected fromautoimmune, inflammatory, proliferative, and hyperproliferative diseasesand immunologically-mediated diseases including rejection oftransplanted organs or tissues and Acquired Immunodeficiency Syndrome(AIDS), wherein said method comprises administering to a patient in needthereof a composition of the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases of therespiratory tract including, without limitation, reversible obstructiveairways diseases including asthma, such as bronchial, allergic,intrinsic, extrinsic and dust asthma, particularly chronic or inveterateasthma (e.g. late asthma airways hyper-responsiveness) and bronchitis.In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including those conditionscharacterized by inflammation of the nasal mucus membrane, includingacute rhinitis, allergic, atrophic thinitis and chronic rhinitisincluding rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta,rhinitis sicca and rhinitis medicamentosa; membranous rhinitis includingcroupous, fibrinous and pseudomembranous rhinitis and scrofoulousrhinitis, seasonal rhinitis including rhinitis nervosa (hay fever) andvasomotor rhinitis, sarcoidosis, farmer's lung and related diseases,fibroid lung, and idiopathic interstitial pneumonia.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases of the boneand joints including, without limitation, rheumatoid arthritis,seronegative spondyloarthropathis (including ankylosing spondylitis,psoriatic arthritis and Reiter's disease), Behcet's disease, Sjogren'ssyndrome, systemic sclerosis, osteoporosis, bone cancer, and bonemetastasis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases and disordersof the skin, including, without limitation, psoriasis, systemicsclerosis, atopical dermatitis, contact dermatitis and other eczematousdermatitis, seborrhoetic dermatitis, lichen planus, pemphigus, bullouspemphigus, epidermolysis bullosa, urticaria, angiodermas, vasculitides,erythemas, cutaneous eosinophilias, uveitis, Alopecia, greata and vernalconjunctivitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including diseases and disordersof the gastrointestinal tract, including, without limitation, celiacdisease, proctitis, eosinophilic gastro-enteritis, mastocytosis,pancreatitis, Crohn's disease, ulcerative colitis, food-relatedallergies which have effects remote from the gut, e.g., migraine,rhinitis and eczema.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including those diseases anddisorders of other tissues and systemic disease, including, withoutlimiation, multiple sclerosis, artherosclerosis, lupus erythematosus,systemic lupus erythematosus, Hashimoto's thyroiditis, myastheniagravis, type I diabetes, nephrotic syndrome, eosinophilia fascitis,hyper IgE syndrome, lepromatous leprosy, sezary syndrome and idiopathicthrombocytopenia purpura, restenosis following angioplasty, tumours (forexample leukemia, lymphomas including prostate cancers), andartherosclerosis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with TEC-kinases including allograft rejectionincluding, without limitation, acute and chronic allograft rejectionfollowing for example transplantation of kidney, heart, liver, lung,bone marrow, skin and cornea; and chronic graft versus host disease.

In some embodiments, the present invention relates to a method oftreating or lessening the severity of one or more of the diseases orconditions associated with TEC-kinases, as recited above, wherein saidmethod comprises administering to a patient in need thereof a compoundor composition according to the present invention.

Bruton's tyrosine kinase (“BTK”), a member of TEC-kinases, is a keysignaling enzyme expressed in all hematopoietic cells types except Tlymphocytes and natural killer cells. BTK plays an essential role in theB-cell signaling pathway linking cell surface B-cell receptor (BCR)stimulation to downstream intracellular responses.

BTK is a key regulator of B-cell development, activation, signaling, andsurvival (Kurosaki, Curr Op Imm, 2000, 276-281; Schaeffer andSchwartzberg, Curr Op Imm 2000, 282-288). In addition, BTK plays a rolein a number of other hematopoietic cell signaling pathways, e.g., Tolllike receptor (TLR) and cytokine receptor-mediated TNF-α production inmacrophages, IgE receptor (Fc epsilon RI) signaling in mast cells,inhibition of Fas/APO-1 apoptotic signaling in B-lineage lymphoid cells,and collagen-stimulated platelet aggregation. See, e.g., C. A. Jeffries,et al., (2003), Journal of Biological Chemistry 278:26258-26264; N. J.Horwood, et al., (2003), The Journal of Experimental Medicine 197:1603-1611; Iwaki et al. (2005), Journal of Biological Chemistry280(48):40261-40270; Vassilev et al. (1999), Journal of BiologicalChemistry 274(3): 1646-1656, and Quek et al. (1998), Current Biology8(20): 1137-1140.

Patients with mutations in BTK have a profound block in B celldevelopment, resulting in the almost complete absence of mature Blymphocytes and plasma cells, severely reduced Ig levels and a profoundinhibition of humoral response to recall antigens (reviewed in Vihinenet al Frontiers in Bioscience 5: d917-928). Mice deficient in BTK alsohave a reduced number of peripheral B cells and greatly decreased serumlevels of IgM and IgG3. BTK deletion in mice has a profound effect on Bcell proliferation induced by anti-IgM, and inhibits immune responses tothymus-independent type II antigens (Ellmeier et al, J Exp Med 192:1611-1623 (2000)). BTK also plays a crucial role in mast cell activationthrough the high-affinity IgE receptor (Fc_epsilon_RI). BTK deficientmurine mast cells have reduced degranulation and decreased production ofproinflammatory cytokines following Fc_epsilon_RI cross-linking(Kawakami et al. Journal of Leukocyte Biology 65: 286-290).

Provided compounds are inhibitors of BTK and are therefore useful fortreating one or more disorders associated with activity of BTK. Thus, insome embodiments, the present invention provides a method for treating aBTK-mediated disorder comprising the step of administering to a patientin need thereof a compound of the present invention, or pharmaceuticallyacceptable composition thereof.

As used herein, the term “BTK-mediated” disorders or conditions as usedherein means any disease or other deleterious condition in which BTK, ora mutant thereof, is known to play a role. Accordingly, anotherembodiment of the present invention relates to treating or lessening theseverity of one or more diseases in which BTK, or a mutant thereof, isknown to play a role. Specifically, the present invention relates to amethod of treating or lessening the severity of a disease or conditionselected from a proliferative disorder or an autoimmune disorder,wherein said method comprises administering to a patient in need thereofa compound or composition according to the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK. In some embodiments, the disease orcondition is an autoimmune disease, e.g., inflammatory bowel disease,arthritis, lupus, rheumatoid arthritis, psoriatic arthritis,osteoarthritis, Still's disease, juvenile arthritis, diabetes,myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis, Graves'disease, Sjogren's syndrome, multiple sclerosis, Guillain-Barresyndrome, acute disseminated encephalomyelitis, Addison's disease,opsoclonus-myoclonus syndrome, ankylosing spondylosis, antiphospholipidantibody syndrome, aplastic anemia, autoimmune hepatitis, celiacdisease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura,optic neuritis, scleroderma, primary biliary cirrhosis, Reiter'ssyndrome, Takayasu's arteritis, temporal arteritis, warm autoimmunehemolytic anemia, Wegener's granulomatosis, psoriasis, alopeciauniversalis, Behcet's disease, chronic fatigue, dysautonomia,endometriosis, interstitial cystitis, neuromyotonia, scleroderma, orvulvodynia. In some embodiments, the disease or condition is ahyperproliferative disease or immunologically-mediated diseasesincluding rejection of transplanted organs or tissues and AcquiredImmunodeficiency Syndrome (AIDS, also known as HIV).

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from heteroimmune conditions or diseases, which include, butare not limited to graft versus host disease, transplantation,transfusion, anaphylaxis, allergies (e.g., allergies to plant pollens,latex, drugs, foods, insect poisons, animal hair, animal dander, dustmites, or cockroach calyx), type I hypersensitivity, allergicconjunctivitis, allergic rhinitis, and atopic dermatitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from an inflammatory disease, e.g., asthma, appendicitis,blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis,cholangitis, cholecystitis, colitis, conjunctivitis, cystitis,dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis,endometritis, enteritis, enterocolitis, epicondylitis, epididymitis,fasciitis, fibrositis, gastritis, gastroenteritis, hepatitis,hidradenitis suppurativa, laryngitis, mastitis, meningitis, myelitismyocarditis, myositis, nephritis, oophoritis, orchitis, osteitis,otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis,pleuritis, phlebitis, pneumonitis, pneumonia, proctitis, prostatitis,pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis,tendonitis, tonsillitis, uveitis, vaginitis, vasculitis, or vulvitis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from a cancer. In one embodiment, the cancer is a B-cellproliferative disorder, e.g., diffuse large B cell lymphoma, follicularlymphoma, chronic lymphocytic lymphoma, chronic lymphocytic leukemia,acute lymphatic leukemia, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, splenicmarginal zone lymphoma, multiple myeloma (also known as plasma cellmyeloma), plasmacytoma, extranodal marginal zone B cell lymphoma, nodalmarginal zone B cell lymphoma, mantle cell lymphoma, mediastinal(thymic) large B cell lymphoma, intravascular large B cell lymphoma,primary effusion lymphoma, Burkitt lymphoma/leukemia, or lymphomatoidgranulomatosis. In some embodiments, the cancer is breast cancer orprostate cancer, or cancer of the mast cells (e.g., mastocytoma, mastcell leukemia, mast cell sarcoma, systemic mastocytosis). In oneembodiment, the cancer is bone cancer. In another embodiment, the canceris of other primary origin and has metastasized to the bone.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK including diseases of the bone and jointsincluding, without limitation, rheumatoid arthritis, seronegativespondyloarthropathis (including ankylosing spondylitis, psoriaticarthritis and Reiter's disease), Behcet's disease, Sjogren's syndrome,systemic sclerosis, osteoporosis, bone cancer, and bone metastasis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, wherein the disease or condition isselected from a thromboembolic disorder, e.g., myocardial infarct,angina pectoris, reocclusion after angioplasty, restenosis afterangioplasty, reocclusion after aortocoronary bypass, restenosis afteraortocoronary bypass, stroke, transitory ischemia, a peripheral arterialocclusive disorder, pulmonary embolism, or deep venous thrombosis.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, including infectious and noninfectiousinflammatory events and autoimmune and other inflammatory diseases.These autoimmune and inflammatory diseases, disorders, and syndromesinclude inflammatory pelvic disease, urethritis, skin sunburn,sinusitis, pneumonitis, encephalitis, meningitis, myocarditis,nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis,dermatitis, gingivitis, appendictitis, pancreatitis, cholocystitus,agammaglobulinemia, psoriasis, allergy, Crohn's disease, irritable bowelsyndrome, ulcerative colitis, Sjogren's disease, tissue graft rejection,hyperacute rejection of transplanted organs, asthma, allergic rhinitis,chronic obstructive pulmonary disease (COPD), autoimmune polyglandulardisease (also known as autoimmune polyglandular syndrome), autoimmunealopecia, pernicious anemia, glomerulonephritis, dermatomyositis,multiple sclerosis, scleroderma, vasculitis, autoimmune hemolytic andthrombocytopenic states, Goodpasture's syndrome, athersclerosis,Addison's disease, Parkinson's disease, Alzheimer's disease, Type Idiabetes, septic shock, systemic lupus erythematosus (SLE), rheumatoidarthritis, psoriatic arthritis, juvenile arthritis, osteoarthritis,chronic idiopathic thrombocytopenic purpura, Waldenstrommacroglobulinemia, myasthenia gravis, Hashimoto's thyroiditis, atopicdermatitis, degenerative joint disease, vitiligo, autoimmunehypopituatarism, Guillain-Barre syndrome, Behcet's disease,scleracierma, mycosis fungoides, acute inflammatory responses (such asacute respiratory distress syndrome and ischemia/reperfusion injury),and Graves' disease.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with BTK, selected from rheumatoid arthritis,multiple sclerosis, B-cell chromic lymphocytic leukemia, acutelymphocytic leukemia, hairy cell leukemia, non-Hodgkin's lymphoma,irritable bowel syndrome, Crohn's Disease, lupus and renal transplant.

Interleukin-2 inducible T-cell kinase (“ITK”) is expressed in T cells,mast cells and natural killer cells. It is activated in T cells uponstimulation of the T cell receptor (TCR), and in mast cells uponactivation of the high affinity IgE receptor. Following receptorstimulation in T cells, Lck, a Src tyrosine kinase family member,phosphorylates Y511 in the kinase domain activation loop of ITK (S. D.Heyeck et al., 1997, J. Biol. Chem., 272, 25401-25408). Activated ITK,together with Zap-70 is required for phosphorylation and activation ofPLC-gamma (S. C. Bunnell et al., 2000, J. Biol. Chem., 275, 2219-2230).PLC-gamma catalyzes the formation of inositol 1,4,5-triphosphate anddiacylglycerol, leading to calcium mobilization and PKC activation,respectively. These events activate numerous downstream pathways andlead ultimately to degranulation (mast cells) and cytokine geneexpression (T cells) (Y. Kawakami et al., 1999, J. Leukocyte Biol., 65,286-290).

The role of ITK in T cell activation has been confirmed in ITK knockoutmice. CD4⁺ T cells from ITK knockout mice have a diminishedproliferative response in a mixed lymphocyte reaction or upon Con A oranti-CD3 stimulation. (X. C. Liao and D. R. Littman, 1995, Immunity, 3,757-769). Also, T cells from ITK knockout mice produced little IL-2 uponTCR stimulation resulting in reduced proliferation of these cells. Inanother study, ITK deficient CD4⁺ T cells produced reduced levels ofcytokines including IL-4, IL-5 and IL-13 upon stimulation of the TCR,even after priming with inducing conditions. (D. J. Fowell, 1999,Immunity, 11, 399-409).

The role of ITK in PLC-gamma activation and in calcium mobilization wasalso confirmed in the T cells of these knockout mice, which had severelyimpaired IP₃ generation and no extracellular calcium influx upon TCRstimulation (K. Liu et al., 1998, J. Exp. Med. 187, 1721-1727). Suchstudies support a key role for ITK in activation of T cells and mastcells. Thus an inhibitor of ITK would be of therapeutic benefit indiseases mediated by inappropriate activation of these cells.

It has been well established that T cells play an important role inregulating the immune response (Powrie and Coffman, 1993, ImmunologyToday, 14, 270-274). Indeed, activation of T cells is often theinitiating event in immunological disorders. Following activation of theTCR, there is an influx of calcium that is required for T cellactivation. Upon activation, T cells produce cytokines, including IL-2,4, 5, 9, 10, and 13 leading to T cell proliferation, differentiation,and effector function. Clinical studies with inhibitors of IL-2 haveshown that interference with T cell activation and proliferationeffectively suppresses immune response in vivo (Waldmann, 1993,Immunology Today, 14, 264-270). Accordingly, agents that inhibit Tlymphocyte activation and subsequent cytokine production, aretherapeutically useful for selectively suppressing the immune responsein a patient in need of such immunosuppression.

Mast cells play a critical roll in asthma and allergic disorders byreleasing pro-inflammatory mediators and cytokines. Antigen-mediatedaggregation of Fc.epsilon.RI, the high-affinity receptor for IgE resultsin activation of mast cells (D. B. Corry et al., 1999, Nature, 402,B18-23). This triggers a series of signaling events resulting in therelease of mediators, including histamine, proteases, leukotrienes andcytokines (J. R. Gordon et al., 1990, Immunology Today, 11, 458-464.)These mediators cause increased vascular permeability, mucus production,bronchoconstriction, tissue degradation and inflammation thus playingkey roles in the etiology and symptoms of asthma and allergic disorders.

Published data using ITK knockout mice suggests that in the absence ofITK function, increased numbers of memory T cells are generated (A. T.Miller et al., 2002 The Journal of Immunology, 168, 2163-2172). Onestrategy to improve vaccination methods is to increase the number ofmemory T cells generated (S. M. Kaech et al., Nature Reviews Immunology,2, 251-262). In addition, deletion of ITK in mice results in reduced Tcell receptor (TCR)-induced proliferation and secretion of the cytokinesIL-2, IL-4, IL-5, IL-10 and IFN-y (Schaeffer et al, Science 284; 638-641(1999), Fowell et al, Immunity 11, 399-409 (1999), Schaeffer et al,Nature Immunology 2 (12): 1183-1188 (2001)). The immunological symptomsof allergic asthma are attenuated in ITK−/−mice. Lung inflammation,eosinophil infiltration and mucus production are drastically reduced inITK−/−mice in response to challenge with the allergen OVA (Mueller etal, Journal of Immunology 170: 5056-5063 (2003)). ITK has also beenimplicated in atopic dermatitis. This gene has been reported to be morehighly expressed in peripheral blood T cells from patients with moderateand/or severe atopic dermatitis than in controls or patients with mildatopic dermatitis (Matsumoto et al, International Archives of Allergyand Immunology 129: 327-340 (2002)).

Splenocytes from RLK−/−mice secrete half the IL-2 produced by wild typeanimals in response to TCR engagement (Schaeffer et al, Science 284:638-641 (1999)), while combined deletion of ITK and RLK in mice leads toa profound inhibition of TCR-induced responses including proliferationand production of the cytokines IL-2, IL-4, IL-5 and IFN-γ (Schaeffer etal, Nature Immunology 2 (12): 1183-1188 (2001), Schaeffer et al, Science284: 638-641 (1999)). Intracellular signalling following TCR engagementis effected in ITK/RLK deficient T cells; inositol triphosphateproduction, calcium mobilization, MAP kinase activation, and activationof the transcription factors NFAT and AP-1 are all reduced (Schaeffer etal, Science 284: 638-641 (1999), Schaeffer et al, Nature Immunology 2(12): 11.83-1188 (2001)).

Provided compounds are inhibitors of ITK and are therefore useful fortreating one or more disorders associated with activity of ITK. Thus, insome embodiments, the present invention provides a method for treatingan ITK-mediated disorder comprising the step of administering to apatient in need thereof a compound of the present invention, orpharmaceutically acceptable composition thereof.

As used herein, the term “ITK-mediated” disorders or conditions as usedherein means any disease or other deleterious condition in which ITK, ora mutant thereof, is known to play a role. Accordingly, anotherembodiment of the present invention relates to treating or lessening theseverity of one or more diseases in which ITK, or a mutant thereof, isknown to play a role. Specifically, the present invention relates to amethod of treating or lessening the severity of a disease or conditionselected from a mast cell-mediated condition, a basophil-mediateddisorder, an immune or allergic disorder, wherein said method comprisesadministering to a patient in need thereof a compound or compositionaccording to the present invention.

In some embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with ITK, wherein the disease or condition is animmune disorder, including inflammatory diseases, autoimmune diseases,organ and bone marrow transplant rejection and other disordersassociated with T cell-mediated immune response or mast cell-mediatedimmune response.

In certain embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with ITK, wherein the disease or condition isacute or chronic inflammation, an allergy, contact dermatitis,psoriasis, rheumatoid arthritis, multiple sclerosis, type I diabetes,inflammatory bowel disease, Guillain-Barre syndrome, Crohn's disease,ulcerative colitis, cancer, graft versus host disease (and other formsof organ or bone marrow transplant rejection) or lupus erythematosus.

In certain embodiments, the present invention provides a method fortreating or lessening the severity of one or more diseases andconditions associated with ITK, wherein the disease or condition is amast cell driven conditions, a basophil-mediated disorder, reversibleobstructive airway disease, asthma, rhinitis, chronic obstructivepulmonary disease (COPD), peripheral T-cell lymphomas or HIV [also knownas Acquired Immunodeficiency Syndrome (AIDS)]. Such conditions includethose described in Readinger, et al., PNAS 105: 6684-6689 (2008).

The Janus kinases (JAK) are a family of tyrosine kinases consisting ofJAK1, JAK2, JAK3 and TYK2. The JAKs play a critical role in cytokinesignaling. The down-stream substrates of the JAK family of kinasesinclude the signal transducer and activator of transcription (STAT)proteins. JAK/STAT signaling has been implicated in the mediation ofmany abnormal immune responses such as allergies, asthma, autoimmunediseases such as transplant rejection, rheumatoid arthritis, amyotrophiclateral sclerosis and multiple sclerosis as well as in solid andhematologic malignancies such as leukemias and lymphomas. Thepharmaceutical intervention in the JAK/STAT pathway has been reviewed[Frank Mol. Med. 5: 432-456 (1999) & Seidel, et al, Oncogene 19:2645-2656 (2000)].

JAK1, JAK2, and TYK2 are ubiquitously expressed, while JAK3 ispredominantly expressed in hematopoietic cells. JAK3 binds exclusivelyto the common cytokine receptor gamma chain (yc) and is activated byIL-2, IL-4, IL-7, IL-9, and IL-15.

The proliferation and survival of murine mast cells induced by IL-4 andIL-9 have, in fact, been shown to be dependent on JAK3- and yc-signaling[Suzuki et al, Blood 96: 2172-2180 (2000)].

Cross-linking of the high-affinity immunoglobulin (Ig) E receptors ofsensitized mast cells leads to a release of proinflammatory mediators,including a number of vasoactive cytokines resulting in acute allergic,or immediate (type I) hypersensitivity reactions [Gordon et al, Nature346: 274-276 (1990) & Galli, N. Engl. J. Med., 328: 257-265 (1993)]. Acrucial role for JAK3 in IgE receptor-mediated mast cell responses invitro and in vivo has been established [Malaviya, et al, Biochem.Biophys. Res. Commun. 257: 807-813 (1999)]. In addition, the preventionof type I hypersensitivity reactions, including anaphylaxis, mediated bymast cell-activation through inhibition of JAK3 has also been reported[Malaviya et al, J. Biol. Chem. 274: 27028-27038 (1999)]. Targeting mastcells with JAK3 inhibitors modulated mast cell degranulation in vitroand prevented IgE receptor/antigen-mediated anaphylactic reactions invivo.

A recent study described the successful targeting of JAK3 for immunesuppression and allograft acceptance. The study demonstrated adose-dependent survival of buffalo heart allograft in Wistar Furthrecipients upon administration of inhibitors of JAK3 indicating thepossibility of regulating unwanted immune responses in graft versus hostdisease [Kirken, Transpl. Proc. 33: 3268-3270 (2001)].

IL-4-mediated STAT-phosphorylation has been implicated as the mechanisminvolved in early and late stages of rheumatoid arthritis (RA).Up-regulation of proinflammatory cytokines in RA synovium and synovialfluid is a characteristic of the disease. It has been demonstrated thatIL-4-mediated activation of IL-4/STAT pathway is mediated through theJanus kinases (JAK 1 & 3) and that IL-4-associated JAK kinases areexpressed in the RA synovium [Muller-Ladner, et al, J. Immunol. 164:3894-3901 (2000)].

Familial amyotrophic lateral sclerosis (FALS) is a fatalneurodegenerative disorder affecting about 10% of ALS patients. Thesurvival rates of FALS mice were increased upon treatment with a JAK3specific inhibitor. This confirmed that JAK3 plays a role in FALS[Trieu, et al, Biochem. Biophys. Res. Commun. 267: 22-25 (2000)].

Signal transducer and activator of transcription (STAT) proteins areactivated by, among others, the JAK family kinases. Results form arecent study suggested the possibility of intervention in the JAK/STATsignaling pathway by targeting JAK family kinases with specificinhibitors for the treatment of leukemia [Sudbeck, et al, Clin. CancerRes. 5: 1569-1582 (1999)]. JAK3 specific compounds were shown to inhibitthe clonogenic growth of JAK3-expressing cell lines DAUDI, RAMOS, LC1;19, NALM-6, MOLT-3 and HL-60. Inhibition of JAK3 and TYK 2 abrogatedtyrosine phosphorylation of STAT3, and inhibited cell growth of mycosisfungoides, a form of cutaneous T cell lymphoma.

According to another embodiment, the invention provides a method fortreating or lessening the severity of a JAK3-mediated disease orcondition in a patient comprising the step of administering to saidpatient a composition according to the present invention.

The term “JAK3-mediated disease”, as used herein means any disease orother deleterious condition in which a JAK3 kinase is known to play arole. Accordingly, another embodiment of the present invention relatesto treating or lessening the severity of one or more diseases in whichJAK3 is known to play a role. Specifically, the present inventionrelates to a method of treating or lessening the severity of a diseaseor condition selected from immune responses such as allergic or type Ihypersensitivity reactions, asthma, autoimmune diseases such astransplant rejection, graft versus host disease, rheumatoid arthritis,amyotrophic lateral sclerosis, and multiple sclerosis, neurodegenerativedisorders such as familial amyotrophic lateral sclerosis (FALS), as wellas in solid and hematologic malignancies such as leukemias andlymphomas, wherein said method comprises administering to a patient inneed thereof a composition according to the present invention.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity ofcancer, an autoimmune disorder, a neurodegenerative or neurologicaldisorder, schizophrenia, a bone-related disorder, liver disease, or acardiac disorder. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular agent, its modeof administration, and the like. Compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

Pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method ofinhibiting protein kinase activity in a biological sample comprising thestep of contacting said biological sample with a compound of thisinvention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or amutant thereof, activity in a biological sample comprising the step ofcontacting said biological sample with a compound of this invention, ora composition comprising said compound. In certain embodiments, theinvention relates to a method of irreversibly inhibiting ErbB1, ErbB2,ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or a mutant thereof, activityin a biological sample comprising the step of contacting said biologicalsample with a compound of this invention, or a composition comprisingsaid compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of protein kinase, or a protein kinase selected from ErbB1,ErbB2, ErbB3, ErbB4, a TEC-kinase, and/or JAK3, or a mutant thereof,activity in a biological sample is useful for a variety of purposes thatare known to one of skill in the art. Examples of such purposes include,but are not limited to, blood transfusion, organ-transplantation,biological specimen storage, and biological assays.

Another embodiment of the present invention relates to a method ofinhibiting protein kinase activity in a patient comprising the step ofadministering to said patient a compound of the present invention, or acomposition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting one or more of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase,and/or JAK3, or a mutant thereof, activity in a patient comprising thestep of administering to said patient a compound of the presentinvention, or a composition comprising said compound. According tocertain embodiments, the invention relates to a method of irreversiblyinhibiting one or more of ErbB1, ErbB2, ErbB3, ErbB4, a TEC-kinase,and/or JAK3, or a mutant thereof, activity in a patient comprising thestep of administering to said patient a compound of the presentinvention, or a composition comprising said compound. In otherembodiments, the present invention provides a method for treating adisorder mediated by one or more of ErbB1, ErbB2, ErbB3, ErbB4, aTEC-kinase, and/or JAK3, or a mutant thereof, in a patient in needthereof, comprising the step of administering to said patient a compoundaccording to the present invention or pharmaceutically acceptablecomposition thereof. Such disorders are described in detail herein.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents, which are normally administered to treatthat condition, may also be present in the compositions of thisinvention. As used herein, additional therapeutic agents that arenormally administered to treat a particular disease, or condition, areknown as “appropriate for the disease, or condition, being treated.”

For example, compounds of the present invention, or a pharmaceuticallyacceptable composition thereof, are administered in combination withchemotherapeutic agents to treat proliferative diseases and cancer.Examples of known chemotherapeutic agents include, but are not limitedto, Adriamycin, dexamethasone, vincristine, cyclophosphamide,fluorouracil, topotecan, taxol, interferons, platinum derivatives,taxane (e.g., paclitaxel), vinca alkaloids (e.g., vinblastine),anthracyclines (e.g., doxorubicin), epipodophyllotoxins (e.g.,etoposide), cisplatin, an mTOR inhibitor (e.g., a rapamycin),methotrexate, actinomycin D, dolastatin 10, colchicine, emetine,trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide,amphotericin, alkylating agents (e.g., chlorambucil), 5-fluorouracil,camptothecin, cisplatin, metronidazole, and Gleevec™, among others. Inother embodiments, a compound of the present invention is administeredin combination with a biologic agent, such as Avastin or VECTIBIX.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with an antiproliferative or chemotherapeutic agent selectedfrom any one or more of Abarelix, aldesleukin, Aldesleukin, Alemtuzumab,Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenictrioxide, Asparaginase, Azacitidine, BCG Live, Bevacuzimab,Fluorouracil, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone,Capecitabine, Camptothecin, Carboplatin, Carmustine, Celecoxib,Cetuximab, Chlorambucil, Cladribine, Clofarabine, Cyclophosphamide,Cytarabine, Dactinomycin, Darbepoetin alfa, Daunorubicin, Denileukin,Dexrazoxane, Docetaxel, Doxorubicin (neutral), Doxorubicinhydrochloride, Dromostanolone Propionate, Epirubicin, Epoetin alfa,Erlotinib, Estramustine, Etoposide Phosphate, Etoposide, Exemestane,Filgrastim, floxuridine fludarabine, Fulvestrant, Gefitinib,Gemcitabine, Gemtuzumab, Goserelin Acetate, Histrelin Acetate,Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide, Imatinib Mesylate,Interferon Alfa-2a, Interferon Alfa-2b, Irinotecan, Lenalidomide,Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine,Megestrol Acetate, Melphalan, Mercaptopurine, 6-MP, Mesna, Methotrexate,Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone,Nelarabine, Nofetumomab, Oprelvekin, Oxaliplatin, Paclitaxel,Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim,Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin, PorfimerSodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim,Sorafenib, Streptozocin, Sunitinib Maleate, Talc, Tamoxifen,Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine, 6-TG,Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin,ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine, Vinorelbine,Zoledronate, or Zoledronic acid.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: treatments for Alzheimer'sDisease such as Aricept® and Excelon®; treatments for Parkinson'sDisease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole,bromocriptine, pergolide, trihexephendyl, and amantadine; agents fortreating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex®and Rebif®), Copaxone®, and mitoxantrone; treatments for asthma such asalbuterol and Singulair®; agents for treating schizophrenia such aszyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agentssuch as corticosteroids, TNF blockers, IL-1RA, azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; and agents for treatingimmunodeficiency disorders such as gamma globulin.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with a monoclonal antibody or an siRNA therapeutic.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of formula I,an additional therapeutic agent, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

The amount of both, an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above)) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Preferably,compositions of this invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of an inventive can beadministered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-1,000 μg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention, or pharmaceutical compositions thereof,may also be incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects may beprevented or mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

5. Probe Compounds

In certain aspects, a compound of the present invention may be tetheredto a detectable moiety to form a probe compound. In one aspect, a probecompound of the invention comprises an irreversible protein kinaseinhibitor of formula I, as described herein, a detectable moiety, and atethering moiety that attaches the inhibitor to the detectable moiety.

In some embodiments, such probe compounds of the present inventioncomprise a provided compound of formula I tethered to a detectablemoiety, R¹, by a bivalent tethering moiety, -T-. The tethering moietymay be attached to a compound of formula I via Ring A, Ring B, or R¹.One of ordinary skill in the art will appreciate that when a tetheringmoiety is attached to R¹, R¹ is a bivalent warhead group denoted as R¹.In certain embodiments, a provided probe compound is selected from anyof formula V, VI, or VII:

wherein each of Ring A, Ring B, R¹, m, p, R^(x), R^(y), R^(v), W¹, andW² is as defined above with respect to formula I, and described inclasses and subclasses herein, R¹ is a bivalent warhead group, T is abivalent tethering moiety; and R^(t) is a detectable moiety.

In some embodiments, R^(t) is a detectable moiety selected from aprimary label or a secondary label. In certain embodiments, R^(t) is adetectable moiety selected from a fluorescent label (e.g., a fluorescentdye or a fluorophore), a mass-tag, a chemiluminescent group, achromophore, an electron dense group, or an energy transfer agent.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and “reporter” and relates to any moiety capableof being detected, e.g., primary labels and secondary labels. A presenceof a detectable moiety can be measured using methods for quantifying (inabsolute, approximate or relative terms) the detectable moiety in asystem under study. In some embodiments, such methods are well known toone of ordinary skill in the art and include any methods that quantify areporter moiety (e.g., a label, a dye, a photocrosslinker, a cytotoxiccompound, a drug, an affinity label, a photoaffinity label, a reactivecompound, an antibody or antibody fragment, a biomaterial, ananoparticle, a spin label, a fluorophore, a metal-containing moiety, aradioactive moiety, quantum dot(s), a novel functional group, a groupthat covalently or noncovalently interacts with other molecules, aphotocaged moiety, an actinic radiation excitable moiety, a ligand, aphotoisomerizable moiety, biotin, a biotin analog (e.g., biotinsulfoxide), a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, a redox-active agent, an isotopicallylabeled moiety, a biophysical probe, a phosphorescent group, achemiluminescent group, an electron dense group, a magnetic group, anintercalating group, a chromophore, an energy transfer agent, abiologically active agent, a detectable label, and any combination ofthe above).

Primary labels, such as radioisotopes (e.g., tritium, ³²P, ³³P, ³⁵S,¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I), mass-tags including, but not limitedto, stable isotopes (e.g., ¹³C, ²H, ¹⁷O, ¹⁸O, ¹⁵N, ¹⁹F, and ¹²⁷I),positron emitting isotopes (e.g., ¹¹C, ¹⁸F, ¹³N, ¹²⁴I, and ¹⁵O), andfluorescent labels are signal generating reporter groups which can bedetected without further modifications. Detectable moities may beanalyzed by methods including, but not limited to fluorescence, positronemission tomography, SPECT medical imaging, chemiluminescence,electron-spin resonance, ultraviolet/visible absorbance spectroscopy,mass spectrometry, nuclear magnetic resonance, magnetic resonance, flowcytometry, autoradiography, scintillation counting, phosphoimaging, andelectrochemical methods.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate may include streptavidin-enzyme conjugates.For antigen labels, secondary intermediates may include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” asused herein refer to moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine(ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3,Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, SYTORNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX,Spectrum Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM,LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP(post-activation), FlASH-CCXXCC, Azami Green monomeric, Azami Green,green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, and SuperGlo GFP.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecoticAcid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass-tags. Stable isotopes (e.g., ¹³C, ²H, ¹⁷O, ¹⁸O, and ¹⁵N)may also be used as mass-tags.

The term “chemiluminescent group,” as used herein, refers to a groupwhich emits light as a result of a chemical reaction without theaddition of heat. By way of example, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants likehydrogen peroxide (H₂O₂) in the presence of a base and a metal catalystto produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

The term “dye,” as used herein, refers to a soluble, coloring substancewhich contains a chromophore.

The term “electron dense group,” as used herein, refers to a group whichscatters electrons when irradiated with an electron beam. Such groupsinclude, but are not limited to, ammonium molybdate, bismuth subnitrate,cadmium iodide, carbohydrazide, ferric chloride hexahydrate,hexamethylene tetramine, indium trichloride anhydrous, lanthanumnitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate,periodic acid, phosphomolybdic acid, phosphotungstic acid, potassiumferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate,silver proteinate (Ag Assay: 8.0-8.5%) “Strong”, silvertetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate,thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranylnitrate, and vanadyl sulfate.

The term “energy transfer agent,” as used herein, refers to a moleculewhich either donates or accepts energy from another molecule. By way ofexample only, fluorescence resonance energy transfer (FRET) is adipole-dipole coupling process by which the excited-state energy of afluorescence donor molecule is non-radiatively transferred to anunexcited acceptor molecule which then fluorescently emits the donatedenergy at a longer wavelength.

The term “moiety incorporating a heavy atom,” as used herein, refers toa group which incorporates an ion of atom which is usually heavier thancarbon. In some embodiments, such ions or atoms include, but are notlimited to, silicon, tungsten, gold, lead, and uranium.

The term “photoaffinity label,” as used herein, refers to a label with agroup, which, upon exposure to light, forms a linkage with a moleculefor which the label has an affinity.

The term “photocaged moiety,” as used herein, refers to a group which,upon illumination at certain wavelengths, covalently or non-covalentlybinds other ions or molecules.

The term “photoisomerizable moiety,” as used herein, refers to a groupwherein upon illumination with light changes from one isomeric form toanother.

The term “radioactive moiety,” as used herein, refers to a group whosenuclei spontaneously give off nuclear radiation, such as alpha, beta, orgamma particles; wherein, alpha particles are helium nuclei, betaparticles are electrons, and gamma particles are high energy photons.

The term “spin label,” as used herein, refers to molecules which containan atom or a group of atoms exhibiting an unpaired electron spin (i.e. astable paramagnetic group) that in some embodiments are detected byelectron spin resonance spectroscopy and in other embodiments areattached to another molecule. Such spin-label molecules include, but arenot limited to, nitryl radicals and nitroxides, and in some embodimentsare single spin-labels or double spin-labels.

The term “quantum dots,” as used herein, refers to colloidalsemiconductor nanocrystals that in some embodiments are detected in thenear-infrared and have extremely high quantum yields (i.e., very brightupon modest illumination).

One of ordinary skill in the art will recognize that a detectable moietymay be attached to a provided compound via a suitable substituent. Asused herein, the term “suitable substituent” refers to a moiety that iscapable of covalent attachment to a detectable moiety. Such moieties arewell known to one of ordinary skill in the art and include groupscontaining, e.g., a carboxylate moiety, an amino moiety, a thiol moiety,or a hydroxyl moiety, to name but a few. It will be appreciated thatsuch moieties may be directly attached to a provided compound or via atethering moiety, such as a bivalent saturated or unsaturatedhydrocarbon chain.

In some embodiments, such detectable moieties are attached to a providedcompound via click chemistry. In some embodiments, such moieties areattached via a 1,3-cycloaddition of an azide with an alkyne, optionallyin the presence of a copper catalyst. Methods of using click chemistryare known in the art and include those described by Rostovtsev et al.,Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun et al., BioconjugateChem., 2006, 17, 52-57. In some embodiments, a click ready inhibitormoiety is provided and reacted with a click ready -T-R^(t) moiety. Asused herein, “click ready” refers to a moiety containing an azide oralkyne for use in a click chemistry reaction. In some embodiments, theclick ready inhibitor moiety comprises an azide. In certain embodiments,the click ready -T-R^(t) moiety comprises a strained cyclooctyne for usein a copper-free click chemistry reaction (for example, using methodsdescribed in Baskin et al., Proc. Natl. Acad. Sci. USA 2007, 104,16793-16797).

In certain embodiments, the click ready inhibitor moiety is of formula:

wherein Ring A, Ring B, W, R^(y), R^(x), and m are as defined above withrespect to Formula I and described herein.

In other embodiments, the click ready inhibitor moiety is of formula:

wherein Ring A, Ring B, W, R^(y), R^(x), m and R¹ are as defined abovewith respect to Formula I and described herein.

Exemplary click ready inhibitors include:

In some embodiments, the click ready -T-R^(t) moiety is:

An exemplary reaction in which a click ready inhibitor moiety and aclick ready -T-R^(t) moiety are joined through a [2+3]-cycloaddition isas follows:

In some embodiments, the detectable moiety, R^(t), is selected from alabel, a dye, a photocrosslinker, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, an antibodyor antibody fragment, a biomaterial, a nanoparticle, a spin label, afluorophore, a metal-containing moiety, a radioactive moiety, quantumdot(s), a novel functional group, a group that covalently ornoncovalently interacts with other molecules, a photocaged moiety, anactinic radiation excitable moiety, a ligand, a photoisomerizablemoiety, biotin, a biotin analog (e.g., biotin sulfoxide), a moietyincorporating a heavy atom, a chemically cleavable group, aphotocleavable group, a redox-active agent, an isotopically labeledmoiety, a biophysical probe, a phosphorescent group, a chemiluminescentgroup, an electron dense group, a magnetic group, an intercalatinggroup, a chromophore, an energy transfer agent, a biologically activeagent, a detectable label, or a combination thereof.

In some embodiments, R^(t) is biotin or an analog thereof. In certainembodiments, R^(t) is biotin. In some embodiments, R^(t) is biotinsulfoxide.

In another embodiment, R^(t) is a fluorophore. In a further embodiment,the fluorophore is selected from Alexa Fluor dyes (Alexa Fluor 350,Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680),AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR,BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665),Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, CascadeYellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl,Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, SYTORNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX,Spectrum Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM,LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP(post-activation), FlASH-CCXXCC, Azami Green monomeric, Azami Green,green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, or SuperGlo GFP.

As described generally above, a provided probe compound comprises atethering moiety, -T-, that attaches the irreversible inhibitor to thedetectable moiety. As used herein, the term “tether” or “tetheringmoiety” refers to any bivalent chemical spacer including, but notlimited to, a covalent bond, a polymer, a water soluble polymer,optionally substituted alkyl, optionally substituted heteroalkyl,optionally substituted heterocycloalkyl, optionally substitutedcycloalkyl, optionally substituted heterocyclyl, optionally substitutedheterocycloalkylalkyl, optionally substituted heterocycloalkylalkenyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycloalkylalkenylalkyl, an optionallysubstituted amide moiety, an ether moiety, an ketone moiety, an estermoiety, an optionally substituted carbamate moiety, an optionallysubstituted hydrazone moiety, an optionally substituted hydrazinemoiety, an optionally substituted oxime moiety, a disulfide moiety, anoptionally substituted imine moiety, an optionally substitutedsulfonamide moiety, a sulfone moiety, a sulfoxide moiety, a thioethermoiety, or any combination thereof.

In some embodiments, the tethering moiety, -T-, is selected from acovalent bond, a polymer, a water soluble polymer, optionallysubstituted alkyl, optionally substituted heteroalkyl, optionallysubstituted heterocycloalkyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkylalkyl, optionally substitutedheterocycloalkylalkenyl, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substitutedheterocycloalkylalkenylalkyl. In some embodiments, the tethering moietyis an optionally substituted heterocycle. In other embodiments, theheterocycle is selected from aziridine, oxirane, episulfide, azetidine,oxetane, pyrroline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine,pyrazole, pyrrole, imidazole, triazole, tetrazole, oxazole, isoxazole,oxirene, thiazole, isothiazole, dithiolane, furan, thiophene,piperidine, tetrahydropyran, thiane, pyridine, pyran, thiapyrane,pyridazine, pyrimidine, pyrazine, piperazine, oxazine, thiazine,dithiane, and dioxane. In some embodiments, the heterocycle ispiperazine. In further embodiments, the tethering moiety is optionallysubstituted with halogen, —CN, —OH, —NO₂, alkyl, S(O), and S(O)₂. Inother embodiments, the water soluble polymer is a PEG group.

In other embodiments, the tethering moiety provides sufficient spatialseparation between the detectable moiety and the protein kinaseinhibitor moiety. In further embodiments, the tethering moiety isstable. In yet a further embodiment, the tethering moiety does notsubstantially affect the response of the detectable moiety. In otherembodiments, the tethering moiety provides chemical stability to theprobe compound. In further embodiments, the tethering moiety providessufficient solubility to the probe compound.

In some embodiments, a tethering moiety, -T-, such as a water solublepolymer is coupled at one end to a provided irreversible inhibitor andto a detectable moiety, R^(t), at the other end. In other embodiments, awater soluble polymer is coupled via a functional group or substituentof the provided irreversible inhibitor. In further embodiments, a watersoluble polymer is coupled via a functional group or substituent of thereporter moiety.

In some embodiments, examples of hydrophilic polymers, for use intethering moiety -T-, include, but are not limited to: polyalkyl ethersand alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, polyoxyethylene glycol, the latter is also known aspolyethylene glycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof, hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof, terpolymers thereof, mixturesthereof, and derivatives of the foregoing. In other embodiments, a watersoluble polymer is any structural form including but not limited tolinear, forked or branched. In further embodiments, multifunctionalpolymer derivatives include, but are not limited to, linear polymershaving two termini, each terminus being bonded to a functional groupwhich is the same or different.

In some embodiments, a water polymer comprises a poly(ethylene glycol)moiety. In further embodiments, the molecular weight of the polymer isof a wide range, including but not limited to, between about 100 Da andabout 100,000 Da or more. In yet further embodiments, the molecularweight of the polymer is between about 100 Da and about 100,000 Da,including but not limited to, about 100,000 Da, about 95,000 Da, about90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, 30,000 Da,about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da,about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500Da, about 400 Da, about 300 Da, about 200 Da, and about 100 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand 50,000 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of the polymer is between about 1,000 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 5,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 10,000 Da and 40,000 Da. In someembodiments, the poly(ethylene glycol) molecule is a branched polymer.In further embodiments, the molecular weight of the branched chain PEGis between, about 1,000 Da and about 100,000 Da, including but notlimited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da.In some embodiments, the molecular weight of a branched chain PEG isbetween about 1,000 Da and about 50,000 Da. In some embodiments, themolecular weight of a branched chain PEG is between about 1,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of a branchedchain PEG is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of a branched chain PEG is betweenabout 5,000 Da and about 20,000 Da. The foregoing list for substantiallywater soluble backbones is by no means exhaustive and is merelyillustrative, and in some embodiments, polymeric materials having thequalities described above are suitable for use in methods andcompositions described herein.

One of ordinary skill in the art will appreciate that when -T-R^(t) isattached to a compound of formula I-a or I-b via the R¹ warhead group,then the resulting tethering moiety comprises the R¹ warhead group. Asused herein, the phrase “comprises a warhead group” means that thetethering moiety formed by —R^(1′)-T- of formula V-a or V-b is eithersubstituted with a warhead group or has such a warhead groupincorporated within the tethering moiety. For example, the tetheringmoiety formed by —R^(1′)-T- may be substituted with an -L-Y warheadgroup, wherein such groups are as described herein. Alternatively, thetethering moiety formed by —R^(1′)-T- has the appropriate features of awarhead group incorporated within the tethering moiety. For example, thetethering moiety formed by —R^(1′)-T- may include one or more units ofunsaturation and optional substituents and/or heteroatoms which, incombination, result in a moiety that is capable of covalently modifyinga protein kinase in accordance with the present invention. Such —R¹-T-tethering moiety are depicted below.

In some embodiments, a methylene unit of an —R^(1′)-T- tethering moietyis replaced by a bivalent -L-Y′- moiety to provide a compound of formulaV-c:

wherein each of Ring A, Ring B, m, p, R^(x), R^(y), R^(v), W¹, W², T, L,Y′, and R^(t) is as defined above and described in classes andsubclasses herein and Y′ is a bivalent version of the Y group definedabove and described in classes and subclasses herein.

In some embodiments, a methylene unit of an —R^(1′)-T- tethering moietyis replaced by an -L(Y)— moiety to provide a compound of formula V-d:

wherein each of Ring A, Ring B, m, p, R^(x), R^(y), R^(v), W¹, W², T, L,Y, and R^(t) is as defined above and described in classes and subclassesherein.

In some embodiments, a tethering moiety is substituted with an L-Ymoiety to provide a compound of formula V-e:

wherein each of Ring A, Ring B, m, p, R^(x), R^(y), R^(v), W¹, W², T, L,Y, and R^(t) is as defined above and described in classes and subclassesherein.

In certain embodiments, the tethering moiety, -T-, has the followingstructure:

In some embodiments, the tethering moiety, -T-, has the followingstructure:

In other embodiments, the tethering moiety, -T-, has the followingstructure:

In certain other embodiments, the tethering moiety, -T-, has thefollowing structure:

In yet other embodiments, the tethering moiety, -T-, has the followingstructure:

In some embodiments, the tethering moiety, -T-, has the followingstructure:

In some embodiments, -T-R^(t) is of the following structure:

In other embodiments, -T-R^(t) is of the following structure:

In certain embodiments, -T-R^(t) is of the following structure:

In some embodiments, a probe compound of formula V, VI, or VII isderived from any compound of Table 5.

In certain embodiments, the probe compound is one of the followingstructures:

It will be appreciated that many -T-R^(t) reagents are commerciallyavailable. For example, numerous biotinylating reagents are availablefrom, e.g., Thermo Scientific having varying tether lengths. Suchreagents include NHS-PEG₄-Biotin and NHS-PEG₁₂-Biotin.

In some embodiments, analogous probe structures to the ones exemplifiedabove are prepared using click-ready inhibitor moieties and click-ready-T-R^(t) moieties, as described herein.

In some embodiments, a provided probe compound covalently modifies aphosphorylated conformation of a protein kinase. In one aspect, thephosphorylated conformation of the protein kinase is either an active orinactive form of the protein kinase. In certain embodiments, thephosphorylated conformation of the protein kinase is an active form ofsaid kinase. In certain embodiments, the probe compound is cellpermeable.

In some embodiments, the present invention provides a method fordetermining occupancy of a protein kinase by a provided irreversibleinhibitor (i.e., a compound of formula I) in a patient, comprisingproviding one or more tissues, cell types, or a lysate thereof, obtainedfrom a patient administered at least one dose of a compound of saidirreversible inhibitor, contacting said tissue, cell type or lysatethereof with a probe compound (i.e., a compound of formula V, VI, orVII) to covalent modify at least one protein kinase present in saidlysate, and measuring the amount of said protein kinase covalentlymodified by the probe compound to determine occupancy of said proteinkinase by said compound of formula I as compared to occupancy of saidprotein kinase by said probe compound. In certain embodiments, themethod further comprises the step of adjusting the dose of the compoundof formula I to increase occupancy of the protein kinase. In certainother embodiments, the method further comprises the step of adjustingthe dose of the compound of formula I to decrease occupancy of theprotein kinase.

As used herein, the terms “occupancy” or “occupy” refer to the extent towhich a protein kinase is modified by a provided covalent inhibitorcompound. One of ordinary skill in the art would appreciate that it isdesirable to administer the lowest dose possible to achieve the desiredefficacious occupancy of the protein kinase.

In some embodiments, the protein kinase to be modified is BTK. In otherembodiments, the protein kinase to be modified is EGFR. In certainembodiments, the protein kinase is JAK. In certain other embodiments,the protein kinase is one or more of ErbB1, ErbB2, ErbB3, or ErbB4. Inyet other embodiments, the protein kinase is TEK, ITK, or BMX.

In some embodiments, the probe compound comprises the irreversibleinhibitor for which occupancy is being determined.

In some embodiments, the present invention provides a method forassessing the efficacy of a provided irreversible inhibitor in a mammal,comprising administering a provided irreversible inhibitor to themammal, administering a provided probe compound to tissues or cellsisolated from the mammal, or a lysate thereof, measuring the activity ofthe detectable moiety of the probe compound, and comparing the activityof the detectable moiety to a standard.

In other embodiments, the present invention provides a method forassessing the pharmacodynamics of a provided irreversible inhibitor in amammal, comprising administering a provided irreversible inhibitor tothe mammal, administering a probe compound presented herein to one ormore cell types, or a lysate thereof, isolated from the mammal, andmeasuring the activity of the detectable moiety of the probe compound atdifferent time points following the administration of the inhibitor.

In yet other embodiments, the present invention provides a method for invitro labeling of a protein kinase comprising contacting said proteinkinase with a probe compound described herein. In one embodiment, thecontacting step comprises incubating the protein kinase with a probecompound presented herein.

In certain embodiments, the present invention provides a method for invitro labeling of a protein kinase comprising contacting one or morecells or tissues, or a lysate thereof, expressing the protein kinasewith a probe compound described herein.

In certain other embodiments, the present invention provides a methodfor detecting a labeled protein kinase comprising separating proteins,the proteins comprising a protein kinase labeled by probe compounddescribed herein, by electrophoresis and detecting the probe compound byfluorescence.

In some embodiments, the present invention provides a method forassessing the pharmacodynamics of a provided irreversible inhibitor invitro, comprising incubating the provided irreversible inhibitor withthe target protein kinase, adding the probe compound presented herein tothe target protein kinase, and determining the amount of target modifiedby the probe compound.

In certain embodiments, the probe compound is detected by binding toavidin, streptavidin, neutravidin, or captavidin.

In some embodiments, the probe is detected by Western blot. In otherembodiments, the probe is detected by ELISA. In certain embodiments, theprobe is detected by flow cytometry.

In other embodiments, the present invention provides a method forprobing the kinome with irreversible inhibitors comprising incubatingone or more cell types, or a lysate thereof, with a biotinylated probecompound to generate proteins modified with a biotin moiety, digestingthe proteins, capturing with avidin or an analog thereof, and performingmulti-dimensional LC-MS-MS to identify protein kinases modified by theprobe compound and the adduction sites of said kinases.

In certain embodiments, the present invention provides a method formeasuring protein synthesis in cells comprising incubating cells with anirreversible inhibitor of the target protein, forming lysates of thecells at specific time points, and incubating said cell lysates with aninventive probe compound to measure the appearance of free protein overan extended period of time.

In other embodiments, the present invention provides a method fordetermining a dosing schedule in a mammal for maximizing occupancy of atarget protein kinase comprising assaying a one or more cell types, or alysate thereof, isolated from the mammal, (derived from, e.g.,splenocytes, peripheral B cells, whole blood, lymph nodes, intestinaltissue, or other tissues) from a mammal administered a providedirreversible inhibitor of formula I, wherein the assaying step comprisescontacting said one or more tissues, cell types, or a lysate thereof,with a provided probe compound and measuring the amount of proteinkinase covalently modified by the probe compound.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Example 1

Synthesis of (6-chloro-pyrimidin-4-yl)-(3-bromo-phenyl)-amine

A solution of 4,6-dichloropyrimidine (5 g, 33.6 mmol), 3-bromoaniline(5.8 g, 33.7 mmol) and N,N-diisopropylethylamine (DIEA) (5.2 g, 40.2mmol) in ethanol (40 mL) was heated at 80° C. for 16 hr. The reactionmixture was cooled to ambient temperature, diethylether (35 mL) wasadded while the mixture was being stirred. The product was precipitated,filtered, washed with water and dried to afford 5.9 g (62% yield) of alight colored solid. MS (m/z): MH⁺=284, 286, 288.

Synthesis of 3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]-phenylamine

A mixture of (6-chloro-pyrimidin-4-yl)-(3-bromo-phenyl)-amine (300 mg,1.1 mmol) and benzene-1,3-diamine (300 mg, 2.75 mmol) in 3 mL of n-BuOHwas heated in a sealed tube to 150° C. for 16 hr. Solvent was removed byvacuum evaporation and the crude product was purified by flashchromatography on silica gel with EtOAc/DCM solvent system to afford 255mg (65% yield) of the title compound as a yellow solid. MS (m/z):MH⁺=356, 358.

The following compounds were prepared in a manner substantially similarto that described in Scheme 1a and the Examples above:

-   -   (a) 3-Bromoaniline and benzene-1,4-diamine gave        4-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]phenylamine: MS        (m/z): MH⁺=356, 358.    -   (b) 3-Chloro-4-fluoroaniline and benzene-1,3-diamine gave        3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-4). MS (m/z):MH⁺=330, 332.    -   (c) 3-Methylaniline and benzene-1,3-diamine gave        3-[6-(3-methylphenylamino)-pyrimidin-4-ylamino]phenylamine        (IR-5). MS (m/z): MH⁺=292.    -   (d) 3-Chloro-4-(3-fluorophenyl)methoxyaniline and        benzene-1,3-diamine gave        3-(6-[3-chloro-4-(3-fluorophenyl)methoxyphenylamino]-pyrimidin-4-ylamino)        phenylamine (I^(R)-6). MS (m/z): MH⁺=436, 438.    -   (e) 3-Bromoaniline and 3-ethynylaniline gave        N⁴-(3-Bromophenyl)-N⁶-(3-ethynylphenyl)pyrimidine-4,6-diamine        (I-12). MS (M+H⁺) 365, 367; ¹H NMR (400 MHz, d⁶-DMSO) δ 9.73 (s,        1H), 9.60 (s, 1H), 8.67 (s, 1H), 7.95 (t, 1H), 7.76 (s, 1H),        7.50 (d, 1H), 7.46 (d, 1H), 7.55 (t, 1H), 7.40 (t, 1H), 7.20 (d,        1H), 7.10 (d, 1H), 6.30 (s, 1H), 4.25 (s, 1H) ppm.    -   (f) 3-Ethynylaniline gave        N⁴,N⁶-bis(3-ethynylphenyl)pyrimidine-4,6-diamine (I-11). MS        (M+H⁺) 311, 312; ¹H NMR (400 MHz, d⁶-DMSO) δ 9.24 (s, 2H), 8.32        (s, 1H), 7.78 (s, 2H), 7.52 (d, 2H), 7.25 (t, 2H), 7.02 (d, 2H),        6.13 (s, 1H), 4.12 (s, 2H) ppm.    -   (g) 4-Phenoxyaniline and 2,6-diaminopyridine gave        2-[6-(4-phenoxyphenyl)-aminopyrimidin-4-yl]amino-6-aminopyridine        (I^(R)-11) MS (m/z): MH⁺=372, 371.    -   (h) 4-Phenoxyaniline and 1,4-diaminobenzene gave        4-[6-(4-phenoxyphenyl)amino-pyrimidin-4-yl]amino-aminobenzene        (I^(R)-14) MS (m/z): MH⁺=370

Example 2

Although a BOC protecting group is depicted in Scheme 2a above and theensuing schemes below, one of ordinary skill in the art will recognizethat other amine protecting groups are amenable for use in preparingcompounds of the present invention. Accordingly, a variety of amineprotecting groups is contemplated.

Synthesis of tert-butyl 3-(6-chloropyrimidin-4-ylamino)phenylcarbamate

4,6-Dichloropyrimidine (1.5 g, 10 mmol), tert-butyl3-aminophenylcarbamate (2.1 g, 10 mmol) and triethylamine (2.2 g, 20mmol) were mixed in ethanol (20 mL), heated at 80° C. for 16 hr. Thereaction mixture was cooled to RT and solvent was removed in vacuo. Thegummy crude product was mixed with 20 mL of DCM and was stirred at RT togive an off-white solid, which was filtered and dried under vacuum (1.6g, 5 mmol). MS (m/z): MH⁺=321, 323.

Synthesis of tert-butyl3-(6-[N-methyl-N-phenylamino]pyrimidin-4-ylamino)phenyl-carbamate

A mixture of tert-butyl 3-(6-chloropyrimidin-4-ylamino)phenylcarbamate(1.6 g, 5 mmol) and N-methylaniline (1.07 g, 10 mmol) was heated at 120°C. in a sealed tube for 2 hr. The reaction mixture was cooled to RT,mixed with 1 mL of 1N NaOH and 5 mL of DCM, stirred for 30 min, theproduct was filtered and dried under vacuum to give a solid product. MS(m/z): MH⁺=392.

Synthesis of3-[6-(N-methyl-N-phenylamino)-pyrimidin-4-ylamino]phenylamine (I^(R)-8)

A mixture of tert-butyl3-(6-[N-methyl-N-phenylamino]pyrimidin-4-ylamino)phenylcarbamate (1.17g, 3 mmol) and TFA (50% in DCM, 10 mL) was stirred at RT for 4 hr. Thereaction solvent was removed under vacuum. The crude product was mixedwith 1 mL of 2N NaOH and 5 mL of EtOAc, was stirred for 30 min, wasfiltered, and was dried under vacuum to give 0.65 g (75%) of the titlecompound, an off-white solid. (I^(R)-8) MS (m/z): MH⁺=292.

The following compounds were prepared in a manner substantially similarto that described in Scheme 2a:

-   -   (a) 4-Phenoxyphenyl amine gave        3-[6-(4-phenoxyphenylamino)-pyrimidin-4-ylamino]phenylamine.        (I^(R)-7) MS (m/z): MH⁺=370.    -   (b) 3-Chloro-4-(2-pyridyl)methoxyaniline gave        3-{[6-(3-chloro-4-(2-pyridyl)methoxyphenylamino)-pyrimidin-4-ylamino]phenylamine.        (I^(R)-9) MS (m/z): MH⁺=419, 421.    -   (c) 3-Chloro-4-(3-fluorobenzyloxy)aniline gave        3-[6-(3-chloro-4-{3-fluorobenzyloxy}phenylamino)-pyrimidin-4-ylamino]-phenyl        amine. (I^(R)-6) MS (m/z): M⁺=436, 438.    -   (d) Aniline gave        3-[6-(phenylamino)-pyrimidin-4-yl]aminophenylamine. (I^(R)-15)        MS (m/z): MH⁺=278.

Synthesis of3-[6-(4-bromo-2-fluorophenylamino)-pyrimidin-4-yl-amino]-phenylamine(I^(R)-17)

A solution of 4-bromo-2-fluoroaniline (701 mg, 3.69 mmol),diisopropylethylamine (0.70 mL, 4.03 mmol), and 4,6-dichloropyrimidine(500 mg, 3.36 mmol) in EtOH (10 mL) was heated in an 85° C. oil bath for5 d. Flash chromatography (2% MeOH/CHCl₃) of the residue gave 380 mg(37%) of N-(4-bromo-2-fluorophenyl)-6-chloropyrimidin-4-amine as a paleyellow solid. MS (m/z): 304, 302. A suspension ofN-(4-bromo-2-fluorophenyl)-6-chloropyrimidin-4-amine (0.37 g, 1.22 mmol)and tert-butyl 3-aminophenylcarbamate (0.28 g, 1.35 mmol) in n-BuOH (4mL) was heated in an oil bath at 120-130° C. for 6 h. The reactionmixture was cooled and concentrated to give 0.68 g of a brown foam.Flash chromatography gave 0.16 g (28%) tert-butyl3-(6-[4-bromo-2-fluorophenyl]aminopyrimidin-4-yl)aminophenylcarbamate asa white solid. MS (m/z): (M+H) 476, 474. tert-Butyl3-(6-[4-bromo-2-fluorophenyl]aminopyrimidin-4-yl)aminophenylcarbamate(0.15 g, 0.32 mmol) was taken up in 4M HCl in dioxane (5 mL). Afterseveral minutes a white solid began to precipitate. The mixture wasallowed to stand for 3 h and was concentrated via rotary evaporation.Saturated aqueous sodium bicarbonate (5 mL) was added and the mixturewas sonicated for several minutes. The solid was collected byfiltration, was washed with water (5 mL), and was dried overnight undervacuum to give 0.12 g (100%) of3-[6-(4-bromo-2-fluorophenylamino)-pyrimidin-4-yl-amino]-phenylamine(I^(R)-17) as a white powder. MS (m/z): (M+H) 376, 374.

Synthesis of tert-butylN-3-(6-chloropyrimidin-4-ylamino)phenyl-N-methylcarbamate

To a stirring solution of tert-butyl3-(6-chloropyrimidin-4-ylamino)phenyl carbamate (2.000 g, 6.235 mmol) in10 mL of THF at 0° C. under N₂ was added drop-wise a solution of 1.0 Mlithium bis(trimethylsilyl)amide in tert-butyl methyl ether (6.23 mL,6.23 mmol). The light yellow solution was stirred at 0° C. for 30 minthen iodomethane (0.43 mL, 6.892 mmol, 1.1 eq) was added. The solutionwas allowed to slowly warm to room temperature overnight, wasconcentrated, and was then partitioned between EtOAc and saturatedKH₂PO₄ solution. The organic extract was washed with brine solution,dried (MgSO₄), was filtered, was concentrated in vacuo and waschromatographed (silica gel, 2% MeOH in CH₂Cl₂) to give 0.904 g oftert-butyl N-3-(6-chloropyrimidin-4-ylamino)phenyl-N-methylcarbamate asan off-white solid. MS (m/z) M+1=335/337 (100/44%), ¹H NMR (CDCl₃) δ8.48 (s, 1H), 7.49 (bs, 1H), 7.38 (m, 1H), 7.24 (m, 1H), 6.91 (m, 1H),6.71 (bs, 1H), 6.30 (s, 1H), 3.48 (s, 3H), 1.53 (s, 9H).

Synthesis ofN-3-[6-(3-Chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl-N-methylamine(Int-C)

A solution of tert-butylN-3-(6-chloropyrimidin-4-ylamino)phenyl-N-methylcarbamate (0.486 g,1.095 mmol) in 25 mL of CH₂Cl₂ was treated with trifluoroacetic acid (5mL). The solution was stirred at room temperature under N₂ for 2 h, wasconcentrated and was partitioned between CHCl₃ and 10% aq. NH₄OHsolution. The organic extract was washed with brine solution, was dried(MgSO₄), was filtered and was concentrated to give 0.630 g ofN-3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl-N-methylamine(Int-C) as yellow oil. MS (m/z): 344/346 (M+1, 100/68%). TLC (SiO₂, 10%MeOH in CHCl₃): R_(f) 0.44.

Example 3

wherein L′ is a subset of L, as defined herein, such that Y-L′C(O)Clresults in formation of provided compounds wherein R¹ is -L-Y wherein aterminal methylene unit of L is replaced with —NHC(O)—.

Synthesis ofN-{3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]-phenyl}-2-propenamide(I-1)

A solution of 3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]phenylamine(250 mg, 0.7 mmol) and triethylamine (180 mg, 1.75 mmol) in 5 mL of THFwas stirred at RT. Acryloyl chloride (80 mg, 0.9 mmol) was added intothe reaction mixture and it was stirred at RT for 1 h. The solvent wasremoved by vacuum evaporation and the crude product was purified byflash chromatography on silica gel with EtOAc/DCM solvent system toafford 115 mg (40% yield) of the title compound as a light coloredsolid. MS (m/z): MH⁺=410, 412. ¹H NMR (DMSO): 10.15 (s, 1H), 9.36 (s,1H), 9.28 (s, 1H), 8.34 (s, 1H), 8.02 (s, 1H), 8.00 (s, 1H), 7.51-7.11(m, 5H), 6.47 (dd, 1H, J=10.1 Hz, J₂=17.0 Hz), 6.27 (dd, 1H, J=1.9 Hz,J₂=17.0 Hz), 6.20 (s, 1H), 5.76 (dd, 1H, J=10.1 Hz, J₂=1.9 Hz) ppm.

The following compounds were prepared in a manner substantially similarto those described in Schemes 3a:

-   -   (a) 4-[6-(3-Bromophenylamino)-pyrimidin-4-ylamino]phenylamine        and acryoyl chloride gave        N-{4-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]phenyl}-2-propenamide        (I-93). MS (m/z): MH⁺=410, 412. ¹H NMR (DMSO): 10.10 (s, 1H),        9.33 (s, 1H), 9.17 (s, 1H), 8.30 (s, 1H), 8.02 (s, 1H), 7.62 (m,        2H), 7.46 (m, 3H), 7.22 (t, 1H, J=8.0 Hz), 7.10 (d, 1H, J=8.0        Hz), 6.43 (dd, 1H, J₁=10.0 Hz, J₂=17.0 Hz), 6.24 (d, 1H, J=17.0        Hz), 6.13 (s, 1H), 5.73 (d, 1H, J=10.0 Hz) ppm.    -   (b) 3-[6-(3-Bromophenylamino)-pyrimidin-4-ylamino]phenylamine        and propionyl chloride gave        N-{3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]phenyl}-propionamide        (I^(R)-3). MS (m/z): MH⁺=412, 414. ¹H NMR (DMSO): 9.78 (s, 1H),        9.28 (s, 1H), 9.16 (s, 1H), 8.26 (s, 1H), 7.96 (s, 1H), 7.76 (s,        1H), 7.43 (d, 1H, J=8.2 Hz), 7.20 (m, 2H), 7.05 (m, 3H), 6.12        (s, 1H), 2.27 (q, 2H, J=7.6 Hz), 1.03 (t, 3H, J=7.6 Hz) ppm.    -   (c) 3-[6-(3-Bromophenylamino)-pyrimidin-4-ylamino]phenylamine        and (E)-2-butenoylchloride gave        (E)-N-{3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]-phenyl}-2-butenamide        (I-8). MS (m/z): M+H⁺=424, 426. ¹H NMR (DMSO): 9.87 (s, 1H),        9.29 (s, 1H), 9.18 (s, 1H), 8.27 (s, 1H), 7.96 (s, 1H), 7.82 (s,        1H), 7.44 (d, 1H, J=8.2 Hz) 7.20 (m, 4H), 7.05 (dd, 1H, J₁=1.0        Hz, J₂=7.8 Hz), 6.75 (m, 1H), 6.15 (m, 2H), 1.81 (d, 3H, J=7.8        Hz) ppm.    -   (d)        3-[6-(3-Chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-4) and acryoyl chloride gave        N-{3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl}-2-propenamide        (I-2). MS (m/z): MH⁺=384, 386. ¹H NMR (DMSO): 9.29 (s, 1H), 9.20        (s, 1H), 8.27 (s, 1H), 7.93 (m, 1H), 7.86 (s, 1H), 7.41 (m, 1H),        7.25 (m, 5H), 6.42 (dd, 1H, J=10.1 Hz, J₂=17.0 Hz), 6.22 (dd,        1H, J=1.9 Hz, J₂=17.0 Hz), 6.09 (d, 1H, J=0.7 Hz), 5.69 (dd, 1H,        J=10.1 Hz, J₂=1.9 Hz) ppm.    -   (e) 3-[6-(3-Methylphenylamino)-pyrimidin-4-ylamino]phenylamine        and acryolyl chloride gave        N-{3-[6-(3-methylphenylamino)-pyrimidin-4-ylamino]phenyl}-2-propenamide        (I-4). MS (m/z): M+H⁺=346. ¹H NMR (DMSO): 9.11 (s, 1H), 9.00 (s,        1H), 8.21 (s, 1H), 7.86 (d, 1H, J=1.7 Hz), 7.31-7.05 (m, 7H),        6.74 (d, 1H, J=7.6 Hz), 6.41 (dd, 1H, J₁=10.0 Hz, J₂=17.0 Hz),        6.20 (dd, 1H, J=2.0 Hz, J₂=17.0 Hz), 6.14 (s, 1H), 5.69 (dd, 1H,        J₁=10.1 Hz, J₂=2.0 Hz), 2.23 (s, 3H) ppm.    -   (f)        3-{6-[3-Chloro-4-(3-fluorophenyl)methoxyphenylamino]-pyrimidin-4-ylamino}phenylamine        (I^(R)-6) and acryolyl chloride gave        N-{3-[6-(3-chloro-4-(3-fluorophenyl)methoxyphenylamino)-pyrimidin-4-ylamino]phenyl}-2-propenamide        (11-6). MS (m/z): M+H⁺=490, 492. ¹H NMR (DMSO): 9.13 (s, 1H),        9.07 (s, 1H), 8.22 (s, 1H), 7.85 (d, 1H, J=1.5 Hz), 7.40-7.10        (m, 10H), 6.41 (dd, 1H, J₁=10.0 Hz, J₂=17.0 Hz), 6.20 (dd, 1H,        J₁=2.0 Hz, J₂=17.0 Hz), 6.05 (s, 1H), 5.70 (dd, 1H, J₁=10.1 Hz,        J₂=2.0 Hz), 5.14 (s, 2H) ppm.    -   (g) 4-Phenoxyphenyl amine gave        3-[6-(4-phenoxyphenylamino)-pyrimidin-4-ylamino]phenylamine.        (I^(R)-7) and acryolyl chloride gave        N-{3-[6-(4-phenoxyphenylamino)-pyrimidin-4-ylamino]phenyl}-2        propenamide (I-13). MS (m/z): MH⁺=424. ¹H NMR (DMSO): 9.14 (s,        1H), 9.10 (s, 1H), 8.22 (s, 1H), 7.89 (s, 1H), 7.52 (d, 2H,        J=9.0 Hz), 7.35-6.92 (m, 11H), 6.42 (dd, 1H, J₁=10.1 Hz, J₂=16.9        Hz), 6.22 (dd, 1H, J=1.9 Hz, J₂=16.9 Hz), 6.12 (s, 1H), 5.70        (dd, 1H, J=1.9 Hz, J₂=10.1 Hz) ppm.    -   (h)        3-[6-(N-methyl-N-phenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-8) and acryoyl chloride gave        N-{3-[6-(N-methyl-N-phenylamino)-pyrimidin-4-ylamino]phenyl}-2        propenamide (I-15): an off-white solid; 70 mg; 20%; MS (m/z):        MH⁺=346. ¹H NMR (DMSO): 10.02 (s, 1H), 9.00 (s, 1H), 8.25 (s,        1H), 7.85 (s, 1H), 7.45-7.12 (m, 6H), 6.45 (dd, 1H), 6.20 (d,        1H), 5.70 (m, 1H), 3.35 (s, 3H) ppm.    -   (i)        3-{[6-(3-Chloro-4-(2-pyridyl)methoxyphenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-9) and acryloyl chloride gave        N-(3-(6-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)-2-propenamide        (I-16). MS (m/z): MH⁺=473, 475(3:1). ¹H NMR (DMSO): 10.10 (s,        1H), 9.17 (s, 1H), 9.08 (s, 1H), 8.55 (m, 1H), 8.24 (s, 1H),        7.92-7.73 (m, 4H), 7.57 (d, 1H), 7.38-7.06 (m, 5H), 6.45 (dd,        1H), 6.23 (dd, 1H), 6.08 (s, 1H), 5.72 (dd, 1H), 5.20 (s, 2H)        ppm.    -   (j)        3-[6-(3-Chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-4) and 1-cyanocyclopropanecarbonyl chloride gave        N-[3-(6-{3-chloro-4-fluorophenylamino)pyrimidin-4-yl)amino]phenyl-1-cyanocyclopropanecarboxamide        (I-47). MS (m/z): M+1=423/425, ¹H NMR (DMSO-d₆) δ 10.04 (s, 1H),        9.18 (s, 1H), 9.12 (s, 1H), 8.27 (d, 1H), 7.88 (s, 1H), 7.8 (s,        1H), 7.47-7.16 (m, 9H), 6.74 (b, 1H), 6.28 (d, 1H), 6.1 (s, 1H),        5.19 (s, 2H), 3.05 (s, 2H), 2.18 (s, 6H).    -   (k)        3-[6-(3-Chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-4) and chloroacetyl chloride gave        2-chloro-N-{3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]-phenyl}-acetamide        (I-49). MS (ES⁺): (M+1)⁺=406 (100%), (M+3)⁺=408 (75%). ¹H-NMR        (DMSO-d₆, δ 10.31 (s, 1H), 9.37 (s, 1H), 9.30 (s, 1H), 8.33 (s,        1H), 8.00 (m, 1H), 7.87 (s, 1H), 7.47-7.24 (m, 5H), 6:15 (s,        1H), 4.26 (s, 2H).    -   (l)        3-[6-(3-Chloro-4-fluorophenyl)aminopyrimidin-4-yl]aminophenylamine        (I^(R)-4) and 2-chloropropionyl chloride gave        2-chloro-N-[3-(6-{3-chloro-4-fluorophenyl}aminopyrimidin-4-yl)aminophenyl]propionamide        (I-50). MS (ES⁺): (M+1)⁺=420 (100%), (M+3)⁺=422 (75%). ¹H-NMR        (DMSO-d₆, δ 10.32 (s, 1H), 9.37 (s, 1H), 9.28 (s, 1H), 8.33 (s,        1H), 8.00-7.98 (m, 1H), 7.87 (s, 1H), 7.47-7.26 (m, 5H), 6.14        (s, 1H), 4.69 (quartet, 1H), 1.61 (d, 3H).    -   (m)₃-[6-(3-Chloro-4-fluorophenylamino)pyrimidin-4-ylamino]phenylamine        (I^(R)-4) and 1-trifluoromethylcyclopropanecarbonyl chloride        gave        N-[3-(6-{3-chloro-4-fluorophenyl}aminopyrimidin-4-yl)aminophenyl]-1-trifluoromethylcyclopropanecarboxamide        (I-51). MS (ES) (m/z): 466/468 [M+1, 100/45%]. ¹H NMR (DMSO-d₆)        δ 9.61 (m, 1H), 9.07-9.18 (m, 2H), 8.14 (m, 1H), 7.63-7.8 (m,        2H), 7.03-7.21 (m, 5H), 5.94 (bs, 1H), 1.12-1.27 (m, 4H).    -   (n)        N-3-[6-(3-Chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl-N-methylamine        (Int-D) and acryolyl chloride gave        N-3-[6-(3-chloro-4-fluorophenylamino)pyrimidin-4-yl]aminophenyl-N-methyl-2-propenamide        (I-53). MS (m/z): 398/400 (M+1, 100/63%). ¹H NMR (DMSO-d₆) δ        10.32 (s, 1H), 9.21 (s, 1H), 8.33 (s, 1H), 7.99 (bs, 1H),        7.25-7.68 (m, 5H), 7.06 (bs, 1H), 6.41-6.47 (m, 1H), 6.26-6.29        (m, 1H), 5.71-5.80 (m, 2H).    -   (o) 3-[6-(4-Bromo-2-fluorophenyl)aminopyrimidin-4-yl]aminophenyl        amine (I^(R)-17) and acryloyl chloride gave        N-3-[6-(4-bromo-2-fluorophenylamino)pyrimidin-4-yl]aminophenyl]-2-propenamide        (I-55). MS (M+H⁺) 430, 428. ¹H NMR (d⁶-DMSO) δ 10.13 (s, 1H),        9.25 (s, 1H), 9.02 (s, 1H), 8.26 (s, 1H), 7.91 (m, 2H), 7.57 (d,        J=11 Hz, 1H), 7.45-7.15 (m, 4H), 6.46 (dd, J=12 and 10 Hz, 1H),        6.26 (d, J=12 Hz, 1H), 6.23 (s, 1H), 5.75 (d, J=10 Hz, 1H).    -   (p)        2-[6-(4-phenoxyphenyl)amino-pyrimidin-4-yl]amino-6-aminopyridine        (IR-11) and acryloyl chloride gave        N-6-[6-(4-phenoxyphenyl)amino-pyrimidin-4-yl)]aminopyridin-2-ylpropenamide        (I-63) ¹H NMR (DMSO-d₆) 5 ppm: 5.75 (d, J=10.20 Hz, 1H), 6.29        (d, J=17.04 Hz, 1H), 6.62 (dd, J=10.08 & 16.92 Hz, 1H),        6.96-7.00 (m, 4H), 7.07-7.11 (m, 1H), 7.18 (bd, J=3.28 Hz, 1H),        7.33-7.38 (m, 3H), 7.62-7.69 (m, 4H), 8.29 (s, 1H), 9.13 (s,        1H), 9.66 (s, 1H), 10.15 (s, 1H); MS: m/z 425.3 (M+1).    -   (q) 2-[6-(3-methylphenoxy)-pyrimidin-4-yl]amino-6-aminopyridine        (I^(R)-12) and acryloyl chloride gave        N-6-[6-(3-methylphenoxy)-pyrimidin-4-yl)]aminopyridin-2-ylpropenamide        (I-65) ¹H NMR (CDCl₃) δ ppm: 2.40 (s, 3H), 5.86 (d, J=9.44 Hz,        1H), 6.49-6.61 (m, 2H), 6.83 (bs, 1H), 6.95-7.05 (m, 3H), 7.15        (d, J=7.48 Hz, 1H), 7.34 (t, J=15.08 Hz, 2H), 7.54 (bd, J=5.56        Hz, 1H), 8.78 (s, 1H), 10.78 (s, 1H); LCMS: m/z 348.8 (M+1).    -   (r) 2-[6-(4-phenoxyphenoxy)-pyrimidin-4-yl]amino-6-aminopyridine        (IR-13) and acryloyl chloride gave        N-6-[6-(4-phenoxyphenoxy)-pyrimidin-4-yl)]aminopyridin-2-yl)propenamide        (I-66) ¹H NMR (MeOD) δ ppm: 5.92 (dd, J=11.60, Hz, 1H),        6.50-6.54 (m, 2H), 6.75-7.28 (m, 10H), 7.37-7.41 (m, 2H), 7.91        (t, J=16.08 Hz, 1H), 8.63 (s, 1H); LCMS: m/z 426 (M+1).    -   (s) 3-[6-(phenylamino)-pyrimidin-4-ylamino]phenylamine. (IR-15)        acryloyl chloride gave        N-3-[6-(phenylamino)-pyrimidin-4-yl]aminophenylpropenamide        (I-67) ¹H NMR (DMSO-d₆) δ ppm: 5.72 (dd, J=2.04 & 10.04 Hz, 1H),        6.19 (s, 1H), 6.25 (dd, J=2 & 16.92 Hz, 1H), 6.46 (dd, J=10.04 &        16.88 Hz, 1H), 6.96 (t, J=7.36 Hz, 1H), 7.19-7.31 (m, 4H), 7.54        (d, J=7.68 Hz, 2H), 7.91 (s, 1H), 8.26 (s, 1H), 9.13 (s, 1H),        9.18 (s, 1H), 10.11 (s, 1H); MS: m/z 332.8 (M+1).

Example 4

Synthesis of(E)-N-(3-(6-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-pyrimidin-4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide(I-19)

3-{[6-(3-chloro-4-(2-pyridyl)methoxyphenylamino)-pyrimidin-4-ylamino]phenylamine(I^(R)-9) was dissolved in N-methylpyrrolidinone (1.2 mL) and addeddropwise over 10 minutes to the ice-cold solution of(E)-4-(dimethylamino)but-2-enoyl chloride hydrochloride in acetonitrile.The reaction was stirred in an ice bath for 2 hr. To the mixture wasadded sodium bicarbonate to pH greater than 9. The oil that formed wasextracted with EtOAc (3×25 mL). A portion of the material was insolubleand set aside. The organic layer was dried (MgSO₄) filtered andevaporated to a dark red oil. Both oils contained substantial amount ofproduct as shown by TLC: SiO₂CHCl₃:MeOH/NH₄OH (8:1) 19:1. The oils werecombined and purified by flash column chromatography silica gel (25×300mm) eluted first with 5% methanol/ammonium hydroxide 8/1 to removenon-polar impurities followed by 10% methanol/ammonium hydroxide 8/1 toelute 59 mg of product. The sample was further purified by a secondflash column chromatography silica gel (25×250 mm) eluted with 10%methanol/ammonium hydroxide 8/1 to give the title compound (16.3 mg,0.03 mmol, 6.4% yield). MS (ES+) 530(M+): 552, (M+Na); ¹H NMR (DMSO-d₆,500 MHz) δ (ppm): 10.04 (s, 1H), 9.19 (s, 1H), 9.14 (s, 1H), 8.60 (s,1H), 8.27 (s, 1H), 7.88 (s, 2H), 7.57 (s, 1H), 7.36 (d, 1H, J=7.4 Hz),7.29 (d, 2H, J=7.8 Hz), 7.22 (d, 2H, J=7.9 Hz), 7.18 (m, 2H), 6.74 (m,1H), 6.30 (d, 1H, J=15 Hz) 6.10 (s, 1H), 5.24 (s, 2H), 3.06 (s, 2H),2.18 (s, 6H) ppm; HPLC: t_(R)=5.15 min, 97.5% (YMC-Pack ODS-A 4.6×100mm, 80% water/20% acetonitrile to 5% water/95% acetonitrile over 5.5min, hold to 9 min.).

Synthesis of(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-bromo-2-butenamide(Int-D)

To a stirring solution of 4-bromo-but-2-enoic acid (0.72 g) at 0° C.under nitrogen atmosphere and triethylamine (0.61 mL, 4.38 mmol) in 5 mLof THF was added iso-butyl chloroformate (0.56 mL, 4.32 mmol). Themixture was stirred for 15 min followed by dropwise addition of asolution ofN-(3-amino-phenyl)-N′-3-methylphenylamino-pyrimidine-4,6-diamine (1.015g, 3.483 mmol) in 50 mL of THF. The reaction was allowed to warm to roomtemperature overnight. The sample was concentrated then partitionedbetween EtOAc and sat. NaHCO₃ solution. The organic extract was washedwith brine solution, dried (MgSO₄), filtered and was concentrated. Theresultant brown foamy solid was washed with diethyl ether and vacuumdried to give 0.960 g of crude(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-bromo-2-butenamide(Int-D) as brick-brown solid. MS (m/z): (M+1) 438/440 (71/75%).

Synthesis of(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-(methyl-prop-2-ynyl)amino-2-butenamide(I-58)

To a stirring solution at 0° C. under N₂ ofN-[3-(6-{3-methylphenyl}amino-pyrimidin-4-ylamino)-4-bromo-2-butenamide(Int-D) (0.492 g, 1.122 mmol) and triethylamine (0.20 mL, 1.44 mmol) in10 mL of THF was added (via syringe) N-methyl-propargylamine (0.11 mL,1.173 mmol). The solution was allowed to warm to room temperatureovernight, was concentrated and was then partitioned between EtOAc andsat. NaHCO₃ solution. The organic extract was washed with brinesolution, was dried (MgSO₄), was filtered and was concentrated. Theresidue was chromatographed (silica gel, 10% MeOH in CHCl₃) to give0.110 g of(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-(methyl-prop-2-ynyl)amino-2-butenamide(I-58). MS (APCI) m/z 427 (M+1, 100%). ¹H NMR (DMSO-d₆) δ 10.06 (s, 1H),9.07 (s, 1H), 9.17 (s, 1H), 8.27 (s, 1H), 7.90 (s, 1H), 7.16-7.36 (m,6H), 6.70-6.73 (m, 1H), 6.79 (d, 1H), 6.20 (s, 1H), 6.32 (d, 1H),3.30-3.49 (m), 3.14-3.27 (m, 3H), 2.18-2.39 (m, 7H which containsinglets at δ 2.25 [3H] and δ 2.29 [3H]).

Synthesis of(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-piperazinyl-2-butenamide(I-57)

To a stirring solution at 0° C. under N₂ ofN-[3-(6-{3-methylphenyl}amino-pyrimidin-4-ylamino)-4-bromo-2-butenamide(Int-D) (2.15 g, 4.91 mmol) and triethylamine (0.86 mL, 6.17 mmol) in 10mL of THF was added dropwise a solution of 1-Boc-piperazine (0.92 g,4.91 mmol) in 10 mL of THF. The solution was allowed to warm to roomtemperature overnight, was concentrated and was then partitioned betweenEtOAc and sat. NaHCO₃ solution. The organic extract was washed withbrine solution, was dried (MgSO₄), was filtered and was concentrated togive 2.76 g of4-{3-[3-(6-{3-methylphenyl}amino-pyrimidin-4-ylamino)-phenylcarbamoyl]-allyl}-piperazine-1-carboxylicacid tert-butyl ester as gummy brown solid. This material was dissolvedin 100 mL of CH₂Cl₂. 20 mL of trifluoroacetic acid was added and themixture was stirred at room temperature under N₂ for 2 h. The mixturewas concentrated in vacuo, was basified with sat. NaHCO₃ solution andwas extracted with EtOAc (2×100 ml). The combined organic extract wasdried (MgSO₄), was filtered and was concentrated in vacuo. The residuewas chromatographed (silica gel, 5% MeOH in CHCl_(3 [)500 mL] then 1%NH₄OH-10% MeOH in CHCl₃) to give 0.404 g of(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-piperazinyl-2-butenamide.(I-57) MS (m/z): 444 (M+1, 100%). ¹H NMR (DMSO-d₆) δ 10.03 (s, 1H), 9.17(s, 1H), 9.07 (s, 1H), 8.27 (s, 1H), 7.90 (s, 1H), 7.17-7.36 (m, 6H),6.79-6.80 (d, 1H), 6.72-6.75 (m, 1H), 6.29 (d, 1H), 6.20 (s, 1H),3.08-3.39 (m, containing water and ˜3H), 2.70-2.72 (m, 4H), 2.30-2.42(m, 4H), 2.29 (s, 3H.

The following compounds were prepared in a manner substantially similarto those described in Scheme 4a, above:

-   -   (a)        3-(6-[3-chloro-4-{3-fluorobenzyloxy}]phenylamino-pyrimidin-4-yl)aminophenylamine        (I^(R)-6) and 4-dimethylamino-2-butenoyl chloride gave        (E)-N-3-([6-(3-chloro-4-{3-fluorobenzyloxy}phenylamino)-pyrimidin-4-yl)aminophenyl-4-(dimethylamino)but-2-enamide        (I-46). MS (m/z): M=547, 549(3:1), ¹H-NMR (DMSO-d₆) δ 10.04 (s,        1H), 9.18 (s, 1H), 9.12 (s, 1H), 8.27 (d, 1H), 7.88 (s, 1H), 7.8        (s, 1H), 7.47-7.16 (m, 9H), 6.74 (b, 1H), 6.28 (d, 1H), 6.1 (s,        1H), 5.19 (s, 2H), 3.05 (s, 2H), 2.18 (s, 6H).    -   (b) 3-[6-(3-methylphenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-5) and 4-(dimethylamino)-2-butenoyl chloride gave        (E)-N-{3-[6-(3-methylphenylamino)-pyrimidin-4-ylamino]phenyl}-4-dimethylamino-2-butenamide        (I-17). MS (m/z): MH⁺=403.    -   (c)        3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenylamine        (IR-4) and 4-(dimethylamino)-2-butenoyl chloride gave        (E)-N-{3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl}-4-dimethylamino-2-butenamide        (I-18). MS (M+H⁺) 441, 443; ¹H NMR (400 MHz, d⁶-DMSO) δ 10.01        (s, 1H), 9.30 (s, 1H), 9.20 (s, 1H), 8.28 (s, 1H), 7.95 (dd, J=7        and 3 Hz, 1H), 7.86 (s, 1H), 7.41 (m, 1H), 7.35-7.10 (m, 3H),        6.69 (dt, J=15 and 5 Hz, 1H), 6.26 (d, J=15 Hz, 1H), 6.11 (s,        1H), 3.02 (d, J=5 Hz, 2H), 2.14 (s, 6H) ppm.    -   (d)        N-3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl-N-methylamine        (Int-C) and 4-dimethylamino-2-butenoyl chloride gave        (E)-N-(3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl-N-methyl-4-(dimethylamino)but-2-enamide        (I-48) MS (ES) (m/z): 455/457 (M+1, 37/13%) and 228 (100%). ¹H        NMR (DMSO-d₆) δ 10.22 (s, 1H), 9.20 (s, 1H), 8.33 (s, 1H), 7.99        (bs, 1H), 7.25-7.68 (m, 5H), 7.05 (bs, 1H), 6.73-6.76 (m, 1H),        6.26-6.29 (m, 1H), 5.71 (s, 1H), 3.06 (bs, 2H), 1.99 (s, 6H).    -   (e) 3-[6-(4-phenoxyphenylamino)-pyrimidin-4-yl]aminophenylamine        (I^(R)-7) and 4-dimethylamino-2-butenoyl chloride gave        (E)-N-3-[6-(4-phenoxyphenyl)amino-pyrimidin-4-yl]aminophenyl-4-(dimethylamino)but-2-enamide        (I-62) ¹H NMR (DMSO-d₆) δ ppm: 2.19 (s, 6H), 3.07 (d, J=5.36 Hz,        2H), 6.16 (s, 1H), 6.29 (d, J=15.4 Hz, 1H), 6.68-6.75 (m, 1H),        6.95-6.98 (m, 4H), 7.06-7.10 (m, 1H), 7.20 (dd, J=7.88 & 8.12        Hz, 1H), 7.24 (d, J=8.32 Hz, 2H), 7.36 (dd, J=7.52 & 7.84 Hz,        2H), 7.55 (d, J=8.76 Hz, 2H), 7.90 (s, 1H), 8.24 (s, 1H), 9.15        (d, J=8.84 Hz, 2H), 10.04 (s, 1H); LCMS: m/z 481 (M+1).

Example 5

wherein L′ is a subset of L, as defined herein, such that Y-L′SO₂Clresults in formation of provided compounds wherein R¹ is -L-Y wherein aterminal methylene unit of L is replaced with —NHSO₂—.

Synthesis ofN-{3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl}-ethenesulfonamide(I-3)

A solution of3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenylamine(I^(R)-4) (300 mg, 0.9 mmol) and triethylamine (500 mg, 5 mmol) in 10 mLof THF was stirred at RT. 2-Chloroethanesulfonyl chloride (360 mg, 2.25mmol) was added into the reaction mixture and stirring was continued atRT for 1 hr. The crude product was purified by flash chromatography onsilica gel with EtOAc/heptane solvent system to afford 35 mg (9%) of thetitle compound, a brown colored solid. MS (m/z): MH⁺=420, 422. ¹H NMR(DMSO): 9.92 (s, 1H), 9.29 (s, 1H), 9.21 (s, 1H), 8.26 (s, 1H), 7.92 (m,1H), 7.40-7.22 (m, 4H), 7.14 (m, 1H), 6.20 (m, 2H), 6.05 (m, 3H) ppm.

The following compounds were prepared in a manner substantially similarto Schemes 5a:

-   -   (a)        3-{6-[3-chloro-4-(3-fluorophenyl)methoxyphenylamino]-pyrimidin-4-ylamino}phenylamine        (I^(R)-6) gave        N-{3-[6-(3-chloro-4-(3-fluorophenyl)methoxy-phenylamino)-pyrimidin-4-ylamino]phenyl}-ethenesulfonamide        (I-7). MS (m/z): M+H⁺=526, 528 (2:1). MS (m/z): M+H⁺=490, 492        (2:1). ¹H NMR (DMSO): 9.91 (s, 1H), 9.16 (s, 1H), 9.07 (s, 1H),        8.22 (s, 1H), 7.73 (s, 1H), 7.40-7.10 (m, 9H), 6.70 (m, 2H),        6.12 (d, 1H, J=17.0 Hz), 6.02 (m, 2H), 5.14 (s, 2H) ppm.    -   (b) 3-[6-(3-methylphenylamino)-pyrimidin-4-ylamino]phenylamine        (I^(R)-5) gave        N-{3-[6-(3-methylphenylamino)-pyrimidin-4-ylamino]phenyl}-ethenesulfonamide        (II-5). MS (m/z): M+H⁺=382. ¹H NMR (DMSO): 9.90 (s, 1H), 9.12        (s, 1H), 9.00 (s, 1H), 8.21 (s, 1H), 7.37 (d, 1H, J=1.7 Hz),        7.26 (m, 3H), 7.13 (m, 2H), 6.70 (m, 3H), 6.13 (m, 2H), 6.00 (d,        1H, J=10.0 Hz), 2.24 (s, 3H) ppm.

Example 6

Synthesis of N-(6-chloro-pyrimidin-4-yl)-pyridine-2,6-diamine

A mixture of 2,6-diaminopyridine (1.530 g, 14.020 mmol) and4,6-dichloropyrimidine (2.610 g, 17.519 mmol) in 15 mL of n-butanol in asealed vial was heated at 100° C. for 72 h. The dark brown sample wascooled, was concentrated to remove most of the n-butanol, and was thenpartitioned between EtOAc and sat. NaHCO₃ solution. An emulsion formed,the sample was filtered through a pad of Celite and the layers wereseparated. The organic extract was washed with sat. KH₂PO₄ and brinesolutions, was dried (MgSO₄), was filtered and was concentrated to brownoily-solid. The sample was suspended into 50 mL of CH₂Cl₂, was cooledand was filtered to give 1.017 g ofN-(6-chloro-pyrimidin-4-yl)-pyridine-2,6-diamine as yellow-orange solid.MS (ES) (m/z) 222/224 (M+1, 100/63%). TLC (SiO₂, 50% EtOAc in hexanes):R_(f) 0.33.

Synthesis ofN-(6-amino-pyridin-2-yl)-N′-(3-chloro-4-fluorophenyl)-pyrimidine-4,6-diamine(Int-E)

A mixture of N-(6-chloro-pyrimidin-4-yl)-pyridine-2,6-diamine (1.000 g,4.512 mmol) and 3-chloro-4-fluoroaniline (1.380 mmol) in 10 mL ofn-butanol in a sealed vial was heated at 120° C. for 24 h. The samplewas cooled, was concentrated to remove most of the n-butanol, and wasthen diluted with EtOAc and sat. NaHCO₃ solution. The sample was stirredat room temperature for 30 min and was filtered. The solid was washedwith fresh EtOAc and water and vacuum dried to give 1.063 g ofN-(6-amino-pyridin-2-yl)-N′-(3-chloro-4-fluorophenyl)-pyrimidine-4,6-diamine(Int-E) as tan solid. MS (ES) (m/z) 331/333 (M+1, 100/65%). TLC (SiO₂,10% MeOH in CHCl₃): R_(f) 0.25.

Synthesis of(E)-N-[6-(3-chloro-4-fluorophenylamino)pyrimidin-4-ylaminopyridin-2-yl]-4-(dimethylamino)but-2-enamide(I-52)

To a stirring suspension at 0° C. under N₂ of4-dimethylamino-but-2-enoic acid, hydrochloride (0.500 g, 3.019 mmol) in15 mL of THF containing 5 drops of DMF was added drop-wise (via syringe)oxalyl chloride (0.28 mL, 3.210 mmol). Gas formation startedimmediately. The sample was stirred at 0° C. for ˜30 min, roomtemperature for ˜2 h, re-cooled to 0° C., then treated with drop-wiseaddition of a solution ofN-(6-amino-pyridin-2-yl)-N′-(3-chloro-4-fluoro-phenyl)-pyrimidine-4,6-diamine(Int-E) (0.500 g, 1.512 mmol) in 15 mL of THF and 3 mL of NMP. Theice-bath was removed, the sample was stirred at room temperature for 2 hthen was partitioned between EtOAc and sat. NaHCO₃ solution. The organicextract was washed with brine solution, dried (MgSO₄), filtered andconcentrated to a yellow solid. The solid was suspended into EtOAc (˜25mL), stirred at room temperature for ˜12 h, filtered and vacuum dried togive 0.459 g (69%) of(E)-N-[6-(3-chloro-4-fluorophenylamino)pyrimidin-4-ylaminopyridin-2-yl]-4-(dimethylamino)but-2-enamide(I-52) as light yellow solid. MS (m/z): 442/444 (M+1, 100/37%). ¹H NMR(DMSO-d₆) δ 10.19 (s, 1H), 9.78 (s, 1H), 9.43 (s, 1H), 8.36 (s, 1H),8.05-8.07 (m, 1H), 7.54-7.72 (m, 4H), 7.32-7.35 (m, 1H), 7.10 (bs, 1H),6.79-6.82 (m, 1H), 6.54-6.57 (m, 1H), 3.14 (bs, 2H), 2.23 (bs, 6H).

Example 7

Synthesis ofN-(5-Amino-2-methylphenyl)-N′-(3-chloro-4-fluorophenyl)-pyrimidine-4,6-diamine(Int-F)

A mixture of 4-(3-chloro-4-fluorophenyl)-6-chloropyrimidin-4-ylamine(I^(R)-4) (1.2 g, 4.6 mmol), 2-methyl-5-nitroaniline (0.85 g, 5.5 mmol)and 1 mL of concentrated HCl in 10 mL of n-butanol was heated at 120° C.for 16 h. With stirring, 5 mL of EtOAc was added to the reactionmixture. The light yellow colored product that precipitated was filteredand dried under vacuum to giveN-(3-chloro-4-fluorophenyl)-N-(2-methyl-5-nitrophenyl)pyrimidine-4,6-diamine.MS (APCI) (m/z): 374/376 (M+1). A mixture ofN-(3-chloro-4-fluorophenyl)-N-(2-methyl-5-nitrophenyl)pyrimidine-4,6-diamine(0.55 g, 1.5 mmol) and iron powder (35 mesh, 0.5 g, 5 eq) in 5 mL ofHOAc and 2 mL of MeOH was heated at reflux for 2 h. The solvent wasremoved in vacuo and the dark colored residue was mixed with 150 mL ofCH₂Cl₂ and 15 mL of sat. K₂CO₃ solution and was stirred at roomtemperature for 30 min. The organic layer was dried (MgSO₄), wasconcentrated, and was then purified by flash chromatography (silica gel,MeOH/NH₄OH/CH₂Cl₂) to afford 0.115 g ofN-(5-amino-2-methylphenyl)-N′-(3-chloro-4-fluorophenyl)pyrimidine-4,6-diamineas a light colored solid. (Int-F) MS (m/z): 344/346 (M+1).

In a manner substantially similar to that described above4-(3-chloro-4-fluorophenylamino)-6-chloropyrimidine and2-methoxy-5-nitroaniline gaveN-(5-amino-2-methoxyphenyl)-N′-(3-chloro-4-fluorophenyl)pyrimidine-4,6-diamine.(Int-G) MS (m/z): 360/362 (M+1).

Synthesis of(E)-N-[3-(6-{3-Chloro-4-fluorophenyl}amino-pyrimidin-4-ylamino)-4-methylphenyl]-4-(dimethylamino)but-2-enamide(I-54)

N-(5-amino-2-methylphenyl)-N′-(3-chloro-4-fluorophenyl)pyrimidine-4,6-diamine(Int-F) and 4-dimethylamino-2-butenoyl chloride were combined in amanner similar to that described in Scheme 4a to give(E)-N-[3-(6-(3-chloro-4-fluorophenyl)amino-pyrimidin-4-ylamino)-4-methylphenyl]-4-(dimethylamino)but-2-enamide(I-54). MS (m/z): 455/457 (M+1, 100/39%). ¹H NMR (DMSO-d₆) δ 11.02 (bs,1H), 10.66 (bs, 1H), 9.92 (bs, 1H), 9.39 (bs, 1H), 7.92-7.94 (m, 1H),7.75 (bs, 1H), 7.27-7.56 (m, 4H), 6.80 (m, 1H), 6.54 (d, 1H), 5.89 (bs,1H), 3.92 (bs, 2H), 2.75 (bs, 6H), 2.17 (bs, 3H).

In a manner substantially similar to that described for the synthesis of(I-54)N-(5-Amino-2-methoxyphenyl)-N′-(3-chloro-4-fluorophenyl)pyrimidine-4,6-diamine(Int-G) and 4-dimethylamino-2-butenoyl chloride gave(E)-N-[3-(6-[3-chloro-4-fluorophenyl)-aminopyrimidin-4-ylamino-4-methoxyphenyl]-4-(dimethylamino)but-2-enamide(I-59). MS (m/z): 471/473 (M+1, 100/41%). ¹H NMR (DMSO-d₆) δ 9.97 (s,1H), 9.28 (s, 1H), 8.46 (s, 1H), 8.26 (s, 1H), 7.93-7.98 (m, 2H),7.44-7.51 (m, 2H), 7.30-7.33 (m, 1H), 7.02 (d, 1H), 6.69-6.72 (m, 1H),6.26 (d, 1H), 6.03 (s, 1H), 3.80 (s, 3H), 3.05 (m, 2H), 2.17 (s, 6H).

Example 8 Synthesis of2-[6-(3-methylphenoxy)-pyrimidin-4-yl]-aminopyridine (I^(R)-12)

A mixture of 2,6-diaminopyridine (1.530 g, 14.020 mmol) and4,6-dichloropyrimidine (2.610 g, 17.519 mmol) in 15 mL of n-butanol in asealed vial was heated at 100° C. for 72 h. The dark brown sample wascooled and was concentrated at reduced pressure to remove most of then-butanol. The residue was then partitioned between EtOAc and saturated.NaHCO3 solution. An emulsion formed, the sample was filtered through apad of Celite and the layers were separated. The organic extract waswashed with sat. KH₂PO₄ and brine solutions, dried (MgSO₄), filtered andconcentrated to brown oily-solid. The sample was suspended into ˜50 mLof CH₂Cl₂, cooled and filtered to give 1.017 g (36%) ofN-(6-chloro-pyrimidin-4-yl)-pyridine-2,6-diamine as yellow-orange solid.MS: m/z 222/224 (M+1, 100/63%). To a solution ofN-(6-chloro-pyrimidin-4-yl)-pyridine-2,6-diamine (100 mg, 0.45 mmol) inDMF (1 mL) was added 3-methylphenol (88 mg, 0.8 mmol) and anhydrousK₂CO₃ (93 mg, 0.6 mmol). The reaction mixture was heated at 145° C. for16 h. It was then cooled and DMF was removed under reduced pressure toget a yellow gummy residue. The residue was taken in EtOAc (20 mL), waswashed sequentially with water (5 mL) and brine (5 mL) and was driedover Na₂SO₄. Filtration followed by concentration under reduced pressureafforded a yellow gum which was further purified by columnchromatography (SiO₂, 60-120, EtOAc/hexane, 5/5) to give 100 mg of2-[6-(3-methylphenoxy)-pyrimidin-4-yl]amino-6-aminopyridine (I^(R)-12)as a pale yellow solid. MS: m/z 294 (M+H)

The following compounds were prepared in a manner substantially similarto the procedure described above:

-   -   (a) 4-Phenoxyphenol and 2,6-diaminopyridine gave        2-[6-(4-phenoxyphenoxy)-pyrimidin-4-yl]amino-6-aminopyridine        (I^(R)-13) MS (m/z): MH⁺=372.    -   (b) 4-nitrophenol and 1,3-diaminobenzene gave        3-[6-(4-nitrophenoxy)-pyrimidin-4-yl]amino-aminobenzene        (I^(R)-16) MS (m/z): MH⁺=324

Example 9 Synthesis ofN-3-[6-(3-ethynylphenylamino)-pyrimidin-4-yl]aminophenyl-2-propenamide(I-68)

To a stirred solution of I^(R)-1 (150 mg, 0.42 mmol) in dry DMF (4.0 mL)under N₂ was added Pd(PPh₃)₂Cl₂ (14.7 mg, 0.021 mmol), CuI (3.9 mg,0.021 mmol), PPh₃ (22.07 mg, 0.08 mmol), and diethylamine (94.8 mg, 6.3mmol). The reaction mixture was purged with N₂ for additional 10 min,trimethylsilylacetylene (45.5 mg, 0.46 mmol) was added, and it was thensubjected to microwave irradiation at 120° C. for 30 min. The mixturewas cooled, was diluted with 5 mL water, was filtered through Celite®and was extracted with EtOAc (2×10 mL). The combined EtOAc extract waswashed with water, then with brine and was dried over Na₂SO₄.Concentration under reduced pressure gave a crude product that waspurified by column chromatography (SiO₂, MeOH/CHCl₃, 1/99) to give 100mg of a brown solid. A solution of this material in 2 mL of dry methanolunder nitrogen atmosphere containing anhydrous K₂CO₃ (73.9 mg, 0.53mmol) was stirred at rt for 16 h. The reaction mixture was filtered andthe filtrate was concentrated under reduced pressure to give a residue,which was further purified by column chromatography (SiO₂, 230-400,MeOH/CHCl₃, 1/99) to give 70 mg of a light brown solid. To a stirredsolution of this material in NMP (1.0 mL) at 0° C. was added acryloylchloride (105 mg, 1.16 mmol), and the reaction mixture was stirred at 0°C. for 1 h. The reaction mixture was then quenched with water, wasbasified with 10% NaHCO₃ sol and was extracted with EtOAc. The combinedEtOAc extract was washed sequentially with water and brine, was driedover Na₂SO₄ and was concentrated under reduced pressure. The residueobtained was further purified by preparative HPLC to give 8 mg ofN-3-[6-(3-ethynylphenyl)aminopyrimidin-4-yl]amino-phenyl-2-propenamide(I-68) as an off white solid. ¹H NMR (DMSO-d₆) 8 ppm: 4.15 (s, 1H),5.73-5.76 (m, 1H), 6.19 (s, 1H), 6.25 (dd, J=1.96 & 17.00 Hz, 2H), 6.46(dd, J=10.2 & 16.96 Hz, 1H), 7.05 (d, J=7.64 Hz, 1H), 7.21-7.33 (m, 3H),7.54 (d, J=7.96 Hz, 1H), 7.81 (s, 1H), 7.91 (s, 1H), 8.32 (s, 1H), 9.28(d, J=11.08 Hz, 2H), 10.13 (s, 1H); MS: m/z 356.8 (M+1).

Example 10 Synthesis ofN-3-[6-(4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]amino]-carbonyl]amino)phenoxypyrimidin-4-yl]aminophenylchloroacetamide (I-69)

Step 1:

To a solution of 3-[6-(4-nitrophenoxy)-pyrimidin-4-yl]amino-aminobenzene(I^(R)-16) (650 mg, 2.01 mmol) in THF (10 mL) was added Et₃N (305 mg,3.01 mmol) and (Boc)₂O (525 mg, 2.4 mmol) under N₂ atmosphere. Thereaction mixture was further heated at 60° C. for 16 h. A residue wasobtained after cooling to room temperature and removal of solvent undervacuum. The residue was dissolved in EtOAc (10 mL). The EtOAc extractwas washed sequentially with water (5 mL) and brine (2 mL), was driedover Na₂SO₄. Concentration under reduced pressure followed bypurification by column chromatography (SiO₂, 60-120, CHCl₃/MeOH, 9/1)gave 400 mg of the Boc derivative as a yellow solid.

Step 2:

The material from Step 1 was dissolved in MeOH (8 mL) and 10% Pd/C (40mg) was added under N₂ atmosphere. The reaction mixture was hydrogenatedin a Parr apparatus (H₂, 3 Kg, rt, 16 h). The reaction mixture wasfiltered through Celite® and the solvent was removed under reducedpressure to give 250 mg, of the amine as a yellow solid.

Step 3:

To the material from Step 2 was added a toluene solution of4-chloro-3-trifluoromethylphenylisocyanate prepared by reacting undernitrogen atmosphere at 0° C. 24 mg of 4-chloro-3-trifluoromethylanilinein 10 mL toluene with 0.08 mL of a 20%-solution of phosgene in toluenefollowed by addition of Et₃N (0.07 mL) and at 110° C. for 16 h. Thereaction mixture of the amine and the isocyanate was further heated at110° C. for 4 h and then was quenched with water (1 mL) and wasextracted with EtOAc (2×20 mL). The EtOAc extract was washed with water(5 mL), brine (2 mL) and dried over Na₂SO₄. Filtration followed byconcentration under vacuum offered a residue which was purified by acolumn chromatography (SiO₂, 230-400, hexane/EtOAc, 9/1) to 20 mg of theBoc/urea intermediate as a yellow solid.

Step 4:

To a solution of 45 mg of this intermediate in CH₂Cl₂ (2 mL) at 0° C.was added TFA (0.01 mL) under N₂ atmosphere. The reaction mixturestirred at room temperature for 4 h, was washed sequentially with 10%NaHCO₃ solution (1 mL) and brine (1 mL) and was dried over Na₂SO₄.Concentration under reduced pressure gave 25 mg of the amine/ureaintermediate as a brownish solid.

Step 5:

To a solution of the amine/urea intermediate from Step 4 (45 mg, 0.05mmol), in THF (2 mL) and Et₃N (10 mg, 0.1 mmol) at 0° C. under N₂atmosphere was added chloroacetyl chloride (55 mg, 0.1 mmol). Thereaction mixture was allowed to come to rt and with stirring for 2 h.The reaction mixture was concentrated under reduced pressure and theresidue was partitioned between EtOAc (4 mL) and water (1 mL). The EtOAclayer was separated, was washed with brine (2 mL) and was dried overNa₂SO₄. Filtration followed by concentration under reduced pressure gavea residue which was further purified by column chromatography (SiO₂,230-400, CHCl₃/MeOH, 9/1) to giveN-3-[6-(4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]phenyl]amino-pyrimidin-4-yl]aminophenylchloroacetamide(I-69) as a pale yellow solid. ¹H NMR (MeOD) 8 ppm: 4.19 (s, 2H), 6.61(s, 1H), 7.13 (d, J=6.92 Hz, 2H), 7.14-7.23 (m, 3H), 7.50-7.55 (m, 3H),7.63-7.66 (m, 1H), 7.95 (s, 1H), 8.00 (s, 1H), 8.30 (s, 1H); MS: m/z 593(M+1).

Example 11 Synthesis of(E)-N-3-(6-[3-methylphenylamino]-pyrimidin-4-yl)aminophenyl-4-(4-acetylpiperazin-1yl)-2-butenamide(I-60)

To a stirring solution at 0° C. under N₂ of(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-piperazinyl-2-butenamide(I-57) (0.291 g, 0.655 mmol) and triethylamine (0.14 mL, 1.004 mmol) in10 mL of THF was added (via syringe) acetyl chloride (0.05 mL, 0.70mmol). The sample was allowed to warm to room temperature overnight, wasconcentrated and was then partitioned between EtOAc and sat. NaHCO₃solution. The organic extract was washed with brine solution, was dried(MgSO₄), was filtered, was concentrated and was chromatographed (silicagel, 1% NH₄OH-10% MeOH in CHCl₃) to give 0.0705 g of(E)-N-3-(6-[3-methylphenyl]aminopyrimidin-4-yl)aminophenyl-4-(4-acetylpiperazin-1yl)-2-butenamide(I-60), a white solid. ¹H NMR (DMSO-d₆ δ 2.00 (s, 3H), 2.29 (s, 3H),2.35-2.41 (m, 4H), 3.15-3.16 (m, 2H), 3.31-3.46 (m, containing water and˜4H), 6.19 (s, 1H), 6.32 (d, 1H), 6.73-6.80 (m, 2H), 7.16-7.35 (m, 6H),7.90 (s, 1H), 8.27 (s, 1H), 9.07 (s, 1H), 9.17 (s, 1H) and 10.05 (s,1H); MS: m/z 486 (M+1, 100%).

Example 12 Synthesis of(E)-N-(3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-ylamino]phenyl-N-methyl-4-(dimethyl-d₆-amino)but-2-enamide(I-61)

To a stirring solution of 4-bromo-but-2-enoic acid (0.28 g) at 0° C.under nitrogen atmosphere and triethylamine (0.25 mL) in 3 mL of THF wasadded iso-butyl chloroformate (0.22 mL). The mixture was stirred for 15min followed by dropwise addition of a solution of3-[6-(3-chloro-4-fluorophenyl)aminopyrimidine-4-yl]aminophenylamine(I^(R)-4) (0.46 g) in 50 mL of THF. The reaction was allowed to warm toroom temperature overnight. The sample was concentrated then partitionedbetween EtOAc and sat. NaHCO₃ solution. The organic extract was washedwith brine solution, dried (MgSO₄), filtered and was concentrated. Theresultant brown foamy solid was washed with diethyl ether and vacuumdried to give 0.378 g of(E)-N-3-(6-[3-chloro-4-fluorophenylamino]pyrimidin-4-ylaminophenyl-4-bromo-2-butenamide(Int-E). MS (m/z): 480, 478, 476 (25/100/80%). To a stirring solution at0° C. under N₂ of Int-E (0.378 g, 0.794 mmol) and triethylamine (0.28mL, 2.01 mmol) in 10 mL of THF was added in one portiondimethyl-d₆-amine hydrochloride (0.070 g, 0.799 mmol). The sample wasallowed to warm to room temperature overnight and then was partitionedbetween EtOAc and sat. NaHCO₃ solution. The organic extract was washedwith brine solution, was dried (MgSO₄), was filtered and wasconcentrated. The residue was chromatographed (silica gel, 1% NH₄OH-10%MeOH in CHCl₃) to give 0.0582 g (16%) of(E)-N-(3-[6-(3-chloro-4-fluorophenylamino)-pyrimidin-4-yl]aminophenyl-4-(dimethyl-d₆-amino)but-2-enamide(I-61), a tan solid. ¹H NMR (DMSO-d₆) δ 3.05-3.07 (m, 2H), 6.16 (s, 1H),6.29-6.32 (m, 1H), 6.72-6.75 (m, 1H), 7.21-7.47 (m, 5H), 7.90 (s, 1H),7.92-8.01 (m, 1H), 8.32 (s, 1H), 9.26 (s, 1H), 9.36 (s, 1H) and 10.06(s, 1H); MS: m/z 447/449 (M+1, 100/49%).

Example 13

I-80 may be prepared in a manner substantially similar to that describedin Scheme 4a, above:

4-[6-(4-phenoxyphenyl)amino-pyrimidin-4-yl]amino-aminobenzene (I^(R)-14)and acryloyl chloride gaveN-4-[6-(4-phenoxyphenyl)amino-pyrimidin-4-yl]aminophenylpropenamide(I-80), an off white solid. ¹H NMR (DMSO-d₆) 8 ppm: 5.73 (dd, J=1.6 &10.0 Hz, 1H), 6.08 (d, J=5.6 Hz, 1H), 6.24 (dd, J=2 & 16.8 Hz, 1H), 6.43(dd, J=10 & 16.8 Hz, 1H), 6.95-6.70 (m, 4H), 7.09 (t, J=7.6 Hz, 1H),7.35-7.39 (m, 2H), 7.45-7.49 (m, 2H), 7.55-7.61 (m, 4H), 8.23 (s, 1H),9.08 (s, 1H), 9.1 (s, 1H), 10.1 (s, 1H); LCMS: m/e 423.8 (M+).

Example 14

Synthesis ofN-6-(6-(phenylamino)pyrimidin-4-ylamino)pyridine-2-yl)acrylamide (I-72)

Step-1

A solution of N²-(6-chloropyrimidin-4-yl)pyridine-2,6-diamine (1) (0.25g, 1.1 mmol) and aniline (2) (0.16 g, 1.7 mmol) in n-BuOH (25 mL) washeated at 120° C. for 12 h in a pressure tube. The reaction mixture wascooled, was dissolved in methanol (10 mL) and was concentrated underreduced pressure. The residue was dissolved in ethyl acetate (35 mL) andwas washed successively with 10% sodium bicarbonate solution (20 mL),water (20 mL), and saturated brine (20 mL). The ethyl acetate extractwas dried over Na₂SO₄ and was concentrated under reduced pressure togive a residue, which was triturated with diethyl ether to give (260 mg,83%) N⁴-(6-aminopyridin-2-yl)-N⁶-phenylpyrimidine-4,6-diamine (3) as anoff-white solid.

Step-2

To a stirred solution of 3 (0.2 g, 0.7 mmol) in NMP (10 mL) was addedacryloyl chloride (0.097 g, 1 mmol), drop wise at 0° C. The reactionmixture was allowed to stir at the same temperature for 20 min and thenwarmed to rt for 1.5 h. It was quenched with 10% sodium bicarbonatesolution (4 mL) and was extracted with ethyl acetate (2×35 mL). Thecombined ethyl acetate layer was washed with water (20 mL), saturatedbrine (20 mL), was dried over Na₂SO₄ and was concentrated under reducedpressure. The residue was further purified by column chromatography(SiO₂, 60-120, Petroleum ether/EtOAc:90/10) to giveN-6-(6-(phenylamino)pyrimidin-4-ylamino)pyridine-2-yl)acrylamide (I-72)as a brown solid. ¹H NMR (DMSO-d₆) δ ppm: 5.80 (dd, J=1.84 & 10.12 Hz,1H), 6.31 (dd, J=1.8 & 16.96 Hz, 1H), 6.64 (dd, J=10.08 & 16.88 Hz, 1H),6.96 (t, J=7.32 Hz, 1H), 7.15-7.20 (m, 1H), 7.28 (t, J=7.52 Hz, 2H),7.34 (s, 1H), 7.60-7.70 (m, 4H), 8.30 (s, 1H), 9.08 (s, 1H), 9.66 (s,1H), 10.06 (s, 1H); LCMS: m/e 332.6 (M+).

Example 15

Synthesis of(E)-4-(dimethylamino)-N-(6-(6-(phenylamino)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-73)

To a stirred solution of acetonitrile (20 mL) and DMF (0.05 mL) under N₂was added N,N-dimethylamino crotonic acid hydrochloride (0.47 g, 2.8mmol). After 10 min this solution was cooled to 0-5° C. Oxalyl chloride(0.44 g, 3.5 mmol) was added and the reaction mixture was maintained at0-5° C. for 30 min. It was allowed to warm to rt and stirring wascontinued for 2 h. It was then heated to 40° C. for 5 min and againbrought to rt and stirred for 10 min to get a light greenish coloredsolution of dimethylaminocrotonyl chloride that was used as such fornext step. To a stirred solution ofN⁴-(6-aminopyridin-2-yl)-N⁶-phenylpyrimidine-4,6-diamine (0.2 g, 0.7mmol) in NMP (10 mL) was added dropwise under N₂ atmosphere at 0° C. thesolution of dimethylaminocrotonyl chloride. The reaction mixture wasmaintained at this temperature for 30 min and was warmed to rt andstirred for 2 h. The mixture was quenched with sodium bicarbonatesolution (1 mL) and was extracted with EtOAc (2×35 mL). The combinedethyl acetate extract was washed with water (20 mL) and brine (20 mL),was dried over Na₂SO₄ and was concentrated under reduced pressure. Theresidue was purified by column chromatography (SiO₂, 60-120, product waseluted at 4-6% methanol in chloroform) to give(E)-4-(dimethylamino)-N-(6-(6-(phenylamino)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-73) as a yellowish solid. ¹H NMR (DMSO-d₆) δ ppm: 2.24 (s, 6H), 3.13(d, J=5.76 Hz, 2H), 6.56 (d, J=15.52 Hz, 1H), 6.80 (d, J=15.36 Hz, 1H),6.95 (t, J=7.36 Hz, 1H), 7.11 (t, J=1.16 Hz, 1H), 7.29 (t, J=7.56 Hz,2H), 7.58 (s, 1H), 7.65-7.7 (m, 4H), 8.31 (d, J=2.44 Hz, 1H), 9.23 (s,1H), 9.68 (s, 1H), 10.16 (s, 1H); LCMS: m/e 390.3 (M+1).

Example 16

Synthesis of(E)-4-(dimethylamino)-N-(6-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-64)

A solution of dimethylaminocrotonyl chloride was prepared by reaction ofdimethylaminocrotonic acid hydrochloride (0.36 g, 2.16 mmol) in CH₃CN (4mL) containing DMF (1 drop) with oxalyl chloride (0.34 g, 2.70 mmol)according to the procedure in Example 15a. This acid chloride was addeddrop wise at 0° C. in to a stirred solution ofN⁴-(6-aminopyridin-2-yl)-N⁶-4-phenoxyphenylpyrimidine-4,6-diamine(I^(R)-11) (0.2 g, 0.54 mmol) in NMP (8 mL). The reaction was stirred at0° C. for 1 h, was diluted with EtOAc (5 mL), and was washed with 10%NaHCO₃ (2 mL), water (2 mL) and brine (2 mL). Drying over Na₂SO₄followed by concentration under reduced pressure offered a residue whichwas further purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) to give(E)-4-(dimethylamino)-N-(6-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-64) as a yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 2.18 (s, 6H), 3.05 (d,J=5.2 Hz, 2H), 6.48 (d, J=15.2 Hz, 1H), 6.79 (d, J=6 & 15.6 Hz, 1H),6.96-6.99 (m, 4H), 7.07-7.15 (m, 2H), 7.36 (t, J=7.6 Hz, 2H), 7.42 (s,1H), 7.64-7.7 (m, 4H), 8.30 (s, 1H), 9.12 (s, 1H), 9.69 (s, 1H), 10.07(s, 1H); LCMS: m/e 482.4 (M+1).

Example 17

Synthesis of(E)-4-(dimethylamino)-N-(6-(6-(3-methylphenoxy)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-74) Step 1

To a solution of N²-(6-chloropyrimidin-4-yl)pyridine-2,6-diamine (100mg, 0.45 mmol) in DMF (1 mL) was added 3-methylphenol (88 mg, 0.8 mmol)and anhydrous K₂CO₃ (93 mg, 0.6 mmol). The reaction mixture was heatedat 145° C. for 16 h. It was then cooled and DMF was removed underreduced pressure to get a yellow gummy residue. The residue was taken inEtOAc (20 mL) and washed with water (5 mL), brine (5 mL) and dried overNa₂SO₄. Filtration followed by concentration under reduced pressureafforded a yellow gum which was further purified by columnchromatography (SiO₂, 60-120, EtOAc/hexane, 5/5) to giveN²-(6-(3-methylphenoxy)pyrimidin-4-yl)pyridine-2,6-diamine (I^(R)-12)(100 mg, 75%) as a pale yellow solid.

Step 2

To a solution of I^(R)-12 (75 mg, 0.25 mmol) in NMP (2 mL) was addeddimethylaminocrotonyl chloride (168 mg, 1.02 mmol) at 0° C. The reactionmixture was stirred at room temperature for 1 h and was then quenchedwith NaHCO₃ solution (2 mL). The mixture was extracted with EtOAc (3×5mL) and the combined EtOAc extract was washed with water (5 mL), brine(5 mL) and was dried over Na₂SO₄. Concentration under reduced pressureafforded a yellow oil, which was further purified by columnchromatography (SiO₂, 60-120, Chloroform/Methanol, 9/1) to give(E)-4-(dimethylamino)-N-(6-(6-(3-methylphenoxy)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-74) as a light brown solid. ¹H NMR (CD₃OD) δ ppm: 2.35 (s, 6H), 2.38(s, 3H), 3.25 (dd, J=1.2 & 6.6 Hz, 2H), 6.40 (d, J=13.16 Hz, 1H),6.92-7.01 (m, 3H), 7.12 (t, J=8.12 Hz, 2H), 7.34 (t, J=7.84 Hz, 1H),7.54 (s, 1H), 7.69 (t, J=6.24 Hz, 1H), 7.79 (d, J=8 Hz, 1H), 8.30 (s,1H); LCMS: m/e 404.8 (M+).

Example 18

Synthesis ofN-(3-(6-(3-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)acrylamide(I-75)

Step 1

A solution of 4,6-dichloropyrimidine (0.5 g, 3.3 mmol), 3-phenoxyaniline(0.75 g, 4 mmol) and DIPEA (0.65 g, 5 mmol) in n-butanol (5 mL) wassubjected to microwave irradiation (110° C., 30 min). The reactionmixture was cooled, concentrated under reduced pressure and the residuewas dissolved in EtOAc (10 mL). This solution was washed with water (5mL) and brine (5 mL), and was dried over Na₂SO₄. Concentrated underreduced pressure gave a residue that was purified by columnchromatography (SiO₂, 60-120, hexane/ethylacetate, 8/2) to give6-chloro-N-(3-phenoxyphenyl)pyrimidine-4-amine (0.56 g, 56%) as anoff-white solid.

Step 2

A solution of 6-chloro-N-(3-phenoxyphenyl)pyrimidine-4-amine (0.25 g,0.8 mmol), 1,3-diaminobenzene (0.36 g, 3.3 mmol), n-butanol (10 mL) andconc. HCl (61 mg, 1.6 mmol) was subjected to microwave irradiation (160°C., 15 min). The reaction mixture was cooled, was concentrated underreduced pressure and the residue was taken in EtOAc (10 mL). The EtOAcsolution was washed with water (5 mL) and brine (5 mL), and was driedover Na₂SO₄. Concentration under reduced pressure gave a residue thatwas purified by column chromatography (SiO₂, 60-120, hexane/ethylacetate, 6/4) to giveN⁴-(3-aminophenyl)-N⁶-(3-phenoxyphenyl)pyrimidine-4,6-diamine (0.1 g,32%) as a brown solid.

Step 3

To a stirred solution ofN⁴-(3-aminophenyl)-N⁶-(3-phenoxyphenyl)pyrimidine-4,6-diamine (40 mg,0.1 mmol), Et₃N (0.03 mL, 0.2 mmol) and NMP (0.4 mL) in CH₂Cl₂ (2 mL),at 0° C., was added acryloyl chloride (6) (29 mg, 0.3 mmol). Thereaction mixture was allowed to come to rt and stirred at thistemperature for 3 h. It was washed with 10% NaHCO₃ solution (2 mL),water (2 mL), brine (2 mL) and dried over Na₂SO₄. Filtration followed byconcentration under reduced pressure offered a residue which waspurified by column chromatography (SiO₂, 230-400, chloroform/methanol,9/1) to gaveN-(3-(6-(3-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)acrylamide(I-75) as a light brown solid. ¹H NMR (DMSO-d₆) 5 ppm: 5.73 (dd, J=1.72& 10 Hz, 1H), 6.18 (s, 1H), 6.24 (dd, J=1.92 & 17 Hz, 1H), 6.45 (dd,J=10.04 & 16.88 Hz, 1H), 6.54-6.57 (m, 1H), 7.01-7.05 (m, 2H), 7.11-7.15(dd, J=0.88 & 7.48 Hz, 1H), 7.19-7.32 (s, 4H), 7.35-7.41 (m, 4H), 7.89(s, 1H), 8.26 (s, 1H), 9.2 (s, 1H), 9.25 (s, 1H), 10.26 (s, 1H); LCMS:m/e 423.8 (M+1).

Example 19

Compound I-76 can be prepared according to Scheme 19a by couplingintermediate 1 with 3-bromophenol, followed by boronic acid coupling ofintermediates 3 and 4 to give intermediate 5. Intermediate 5 can then betreated with acryloyl chloride using a protocol similar to that inExample 6 to give compound I-76.

Example 20

Compound I-77 can be prepared according to Scheme 20a by treatment ofintermediate 5 with (E)-4-(dimethylamino)but-2-enoyl chloride using aprotocol similar to that in Example 6.

Example 21

Synthesis ofN-methyl-N-(6-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyridin-2-yl)acrylamide(I-78)

Step 1

To a solution of 2,6-diaminopyridine (10 g, 91.63 mmol) in dry THF (100mL) was added K₂CO₃ (18.8 g, 136.23 mmol) and CH₃I (13 g, 91.63 mmol)and the reaction mixture was stirred at room temperature for 16 h. Waterwas added (10 mL) and the mixture was extracted with EtOAc (100 mL). TheEtOAc layer was dried and was concentrated under reduced pressure. Theresidue was further purified by column chromatography (SiO₂, 60-120,chloroform) to give 2-methylamino-6-aminopyridine (1.1 g, 10%) as abrown solid.

Step 2

A mixture of 2-methylamino-6-aminopyridine (0.5 g, 4.04 mmol),4,6-dichloropyrimidine (1.51 g, 10.13 mmol), DIPEA (1.5 g, 12.17 mmol)in n-butanol (5 mL) was heated at 120° C. for 16 h. The reaction mixturewas cooled, was concentrated under reduced pressure and the residue wastaken in dichloromethane (25 mL). The dichloromethane solution waswashed with NaHCO₃ solution (2 mL), water (2 mL) and brine (2 mL), wasdried over Na₂SO₄ and was concentrated under reduced pressure. Theresidue was purified by column chromatography (SiO₂, 60-120, petroleumether/ethyl acetate, 6/4) to giveN²-(6-chloropyrimidin-4-yl)-N⁶-methylpyridine-2,6,diamine (0.3 g, 33%)as a yellow solid.

Step 3

A solution of N²-(6-chloropyrimidin-4-yl)-N⁶-methylpyridine-2,6,diamine(0.3 g 1.27 mmol), 4-phenoxy aniline (0.28 g, 1.52 mmol) and conc. HCl(2 drops) in n-butanol (2 mL) was subjected to microwave irradiation(120° C., 1 h). The reaction mixture was cooled, was concentrated underreduced pressure, and the residue was diluted with CH₂Cl₂ (5 mL). Thedichloromethane solution was washed with NaHCO₃ (2 mL), water (2 mL),and brine (2 mL), and was dried over Na₂SO₄. Filtration followed byconcentration under reduced pressure gave a residue which was purifiedby column chromatography (SiO₂, 60-120, chloroform/methanol, 9/1) togiveN⁴-(6-(methylamino)pyridine-2-yl)-N⁶-(4-phenoxyphenyl)pyrimidine-4,6-diamine(0.2 g, 41%) as a light brown solid.

Step 4

To a solution ofN⁴-(6-(methylamino)pyridine-2-yl)-N⁶-(4-phenoxyphenyl)pyrimidine-4,6-diamine(0.07 g, 0.18 mmol) in NMP (1 mL) was added acryloyl chloride (0.032 g,0.36 mmol) at 0° C. and the reaction mixture was stirred at rt for 1 h.The reaction mixture was diluted with dichloromethane (2 mL), was washedwith NaHCO₃ (1 mL), water (1 mL), and brine (1 mL). The dichloromethanesolution was dried over Na₂SO₄ and was concentrated under reducedpressure. The residue was purified by column chromatography (SiO₂,60-120, chloroform/methanol, 9/1) to giveN-methyl-N-(6-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyridin-2-yl)acrylamide(I-78) as a yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 3.22 (s, 3H), 5.60(dd, J=2.56 & 9.76 Hz, 1H), 6.12-6.16 (m, 2H), 6.79 (d, J=7.56 Hz, 1H),6.96 (d, J=8.64 Hz, 5H), 7.09 (t, J=7.32 Hz, 1H), 7.23 (s, 1H),7.33-7.37 (m, 4H), 7.45 (d, J=8.2 Hz, 2H), 7.32 (t, J=7.8 Hz, 1H), 8.24(s, 1H); LCMS: m/e 439.3 (M+1).

Example 22

Synthesis of(E)-4-(dimethylamino)-N-methyl-N-(6-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-79)

To a solution of dimethylaminocrotonic acid hydrochloride (0.120 g, 0.72mmol) in CH₃CN (1.4 mL) was added DMF (1 drop) followed by oxalylchloride (0.07 mL, 0.91 mmol) at 0° C. under nitrogen atmosphere. Thereaction was allowed to stir at this temperature for 30 min and then atrt for 2 h. This acid chloride was added, drop wise, at 0° C. in to astirred solution ofN⁴-(6-(methylamino)pyridine-2-yl)-N⁶-(4-phenoxyphenyl)pyrimidine-4,6-diamine(0.07 g, 0.18 mmol) in NMP (2.8 mL). The reaction was allowed to stir at0° C. for 1 h, diluted with EtOAc (5 mL), and washed with 10% NaHCO₃ (2mL), water (2 mL) and brine (2 mL). drying over Na₂SO₄ followed byconcentration under reduced pressure gave a residue which was furtherpurified by column chromatography (SiO₂, 60-120, chloroform/methanol,9/1) to give(E)-4-(dimethylamino)-N-methyl-N-(6-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyridine-2-yl)but-2-enamide(I-79) as a yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 2.05 (s, 6H), 2.91 (d,J=6 Hz, 2H), 3.27 (s, 3H), 6.08 (d, J=15.2 Hz, 1H), 6.65 (dd, J=5.6 &14.8 Hz, 1H), 6.82 (d, J=7.6 Hz, 1H), 6.97-7.00 (m, 4H), 7.10 (t, J=7.2Hz, 1H), 7.20 (s, 1H), 7.35-7.39 (m, 2H), 7.46 (d, J=8 Hz, 1H), 7.53 (d,J=8.8 Hz, 2H), 7.75 (t, J=8 Hz, 1H), 8.30 (s, 1H), 9.30 (s, 1H), 9.95(s, 1H); LCMS: m/e 496 (M+1).

Example 23

Synthesis of(E)-4-dimethylamino)-N-(6-(6-(4-phenoxyphenoxy)pyrimidin-4-ylamino)pyridin-2-yl)but-2-enamide(I-82)

To a stirred solution ofN²-(6-(4-phenoxyphenoxy)pyrimidin-4-yl)pyridine-2,6-diamine (0.65 g,1.75 mmol) in NMP (10 mL) was added dimethylaminocrotonyl chloride(1.026 g, 7 mmol) at 0° C. The reaction mixture was allowed to come toroom temperature and kept at it for 1 h. It was diluted withdichloromethane (10 mL), was washed with NaHCO₃ solution (2 mL) andwater (2 mL), and dried over Na₂SO₄. The dichloromethane solution wasfiltered and was concentrated under reduced pressure to give a residuethat was purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) to give(E)-4-dimethylamino)-N-(6-(6-(4-phenoxyphenoxy)pyrimidin-4-ylamino)pyridin-2-yl)but-2-enamide(I-82) as an off white solid. ¹H NMR (DMSO-d₆) δ ppm: 2.19 (s, 6H), 3.08(d, J=5.52 Hz, 2H), 6.50 (d, J=15.4 Hz, 1H), 6.77 (td, J=5.92 & 15.4 Hz,1H), 7.00-7.07 (m, 5H), 7.15 (t, J=7.36 Hz, 1H), 7.21 (dd, J=2.2 & 8.92Hz, 2H), 7.41 (t, J=7.52 Hz, 2H), 7.68 (t, J=7.96 Hz, 1H), 7.77 (d,J=7.96 Hz, 1H), 7.95 (s, 1H), 8.35 (s, 1H), 10.20 (s, 1H), 10.40 (s,1H); LCMS: m/e 483 (M+1).

Example 24

Synthesis of2-(hydroxy(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)methyl)acrylamide(I-84)

Step 1

To a stirred solution ofN-methoxy-N-methyl-3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzamide(0.75 g, 1.7 mmol) in THF (10 mL) was added LAH (3.4 mL, 3.4 mmol, 1 Msolution in THF) at −60° C. The reaction mixture was stirred at −60° C.for 1 h, was quenched with Na₂SO₄ solution (2 mL) and was extracted withethyl acetate (10 mL). The organic layer was separated and washed withwater (2 mL) and with brine solution (2 mL) and was dried over anhydrousNa₂SO₄. Filtration followed by concentration under reduced pressure gavea residue that was purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) to give3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzaldehyde (0.6 g, 92%)as an off yellow solid.

Step 2

To a stirred solution of3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzaldehyde (100 mg,0.26 mmol) and acrylonitrile (36 mg, 0.52 mmol) in 1,4-dioxane/H₂O (0.5mL/0.5 mL) was added DABCO (29 mg, 0.26 mmol) at rt. Stirring wascontinued at room temperature for 48 h after which time the reactionmixture was concentrated under reduced pressure. The residue obtainedwas further purified by column chromatography (SiO₂, 60-120, petether/ethyl acetate, 6/4) to give2-(hydroxy(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)methyl)acrylamide(I-84) as a white solid. ¹H NMR (DMSO-d₆) δ ppm: 5.33 (s, 1H), 6.06 (s,1H), 6.20 (s, 1H), 6.25 (s, 1H), 6.80 (s, 1H), 6.88 (s, 1H), 6.95-7.05(m, 4H), 7.09-7.13 (m, 2H), 7.16 (bd, J=7.92 Hz, 1H), 7.32-7.39 (m, 5H),7.47 (s, 1H), 8.31 (s, 1H); LCMS: m/e 436 (M+1).

Example 25

Synthesis of1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyrrolidin-1-yl)prop-2-en-1-one(I-85)

Step 1

A solution of 4,5-dichloropyrimidine (0.6 g, 4.02 mmol),3-amino-Boc-pyrrolidine (0.5 g, 2.6 mmol) and DIPEA (1.73 g, 13.3 mmol)in n-butanol (5.0 mL) was heated in a pressure tube (120° C., 12 h). Itwas cooled, quenched with water (10 mL), and was extracted with EtOAc(2×25 mL). The combined EtOAc extract was washed with water (5 mL),brine (5 mL), dried over Na₂SO₄ and concentrated under reduced pressureto afford tert-butyl3-(6-chloropyrimidin-4-ylamino)pyrrolidine-1-carboxylate (0.4 g, 50%) asa yellow solid.

Step 2

To a stirring solution of tert-butyl3-(6-chloropyrimidin-4-ylamino)pyrrolidin-1-carboxylate (0.5 g, 1.6mmol) and 4-phenoxy aniline (0.309 g, 1.6 mmol) in ethanol (4 mL) wasadded acetic acid (0.1 mL) and the reaction mixture was heated at 100°C. for 36 h. The reaction mixture was cooled, ethanol was removed underreduced pressure and the residue was taken in ethyl acetate (10 mL). Itwas washed with NaHCO₃ solution (2 mL), brine (2 mL), dried over Na₂SO₄and concentrated under reduced pressure. The residue was furtherpurified by column chromatography (SiO₂, 60-120, chloroform/methanol,9/1) to yield tert-butyl3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyrrolidine-1-carboxylate(0.3 g, 42.8%) as a white solid.

Step 3

To a stirred solution of tert-butyl3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyrrolidin-1-carboxylate(0.1 g, 0.2 mmol) in dry CH₂Cl₂ (2.0 mL) at 0° C. was added CF₃COOH (2mL, 20 vol.) and the reaction mixture was kept at this temperature for30 min. It was allowed to come to rt and stir at this temperature for 3h. The reaction mixture was concentrated under reduced pressure and theresidue was quenched with water (2 mL), basified with NaHCO₃ solution,and was extracted with ethyl acetate (2×8 mL). The combined ethylacetate extract was washed with water (2 mL) and brine (2 mL), was driedover Na₂SO₄ and was concentrated under reduced pressure to giveN⁴-(4-phenoxyphenyl)-N⁶-(pyrrolidin-3-yl)pyrimidine-4,6-diamine (0.025g, 32.4%) as a light a brown solid.

Step 4

To a stirred solution ofN⁴-(4-phenoxyphenyl)-N⁶-(pyrrolidin-3-yl)pyrimidine-4,6-diamine (0.13 g,0.3 mmol) in THF (1.5 mL) at −60° C. were added DIPEA (0.07 g, 0.5 mmol)and acryloyl chloride (1 M solution in THF, 0.3 mL, 0.3 mmol), and thereaction mixture was stirred at −60° C. for 5 min. The reaction mixturewas quenched by adding water and it was extracted with EtOAc (2×5 mL).The combined EtOAc extract was washed with brine (3 mL), was dried overNa₂SO₄ and was concentrated under reduced pressure. The residue obtainedwas purified by column chromatography (SiO₂, 230-400,chloroform/methanol:98/2) to give1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)pyrrolidin-1-yl)prop-2-en-1-one(I-85) as a light green solid. ¹H NMR (DMSO-d₆) δ ppm: 1.83-1.86 &1.90-1.94 (m, 1H), 2.07-3.0 & 2.16-2.20 (m, 1H), 3.38-3.86 (m, 4H),4.2-4.75 & 4.35-4.5 (bs, 1H), 5.65 (dt, J=2 & 10 Hz, 1H), 5.78 (d, J=3.6Hz, 1H), 6.11 & 6.15 (dd, J=2.4 & 7.0 Hz & dd, J=2.4 & 7.2 Hz, 1H),6.51-6.64 (m, together 1H), 6.93-7.00 (m, 4H), 7.07-7.18 (m, 2H), 7.36(t, J=8 Hz, 2H), 7.51 (d, J=8 Hz, 2H), 8.12 (s, 1H), 8.93 (s, 1H); LCMS:m/e 401.8 (M+1).

Example 26

Synthesis of1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1one(I-86)

Step 1

A solution of 4,6-dichloropyrimidine (0.1 g, 0.671 mmol),3-amino-Boc-piperidine (0.16 g, 0.80 mmol) and DIPEA (0.086 g, 6.71mmol) in n-butanol (5.0 mL) was heated in a pressure tube (120° C., 12h). The solution was cooled, was quenched with water (2 mL) and wasextracted with EtOAc (2×15 mL). The combined EtOAc extract was washedwith water (5 mL), brine (5 mL), was dried over Na₂SO₄ and wasconcentrated under reduced pressure to give tert-butyl3-(6-chloropyrimidin-4-ylamino)piperidine-1-carboxylate, which was driedunder high vacuum and was used as such for next step without furtherpurification.

Step 2

A solution of tert-butyl3-(6-chloropyrimidin-4-ylamino)piperidine-1-carboxylate (0.15 g, 0.48mmol), 4-phenoxyaniline (0.089 g, 0.48 mmol), Pd(OAc)₂ (0.010 g, 0.048mmol), BINAP (0.014 g, 0.024 mmol) and Cs₂CO₃ (0.39 g, 1.2 mmol) indegassed toluene (toluene was purged with N₂ for 15 min) was heated for12 h at 100° C. under N₂ atmosphere. The reaction mixture was cooled,was diluted with EtOAc (20 mL) and was washed with water (4 mL) andbrine (2 mL) and was dried over Na₂SO₄. The crude product obtained waspurified by column chromatography (SiO₂, 230-400, chloroform/methanol:99/1) to give tert-butyl3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)piperidine-1-carboxylate(90 mg, 40.9%) as a light yellow solid.

Step 3

To a stirred solution of tert-butyl3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)piperidine-1-carboxylate(110 mg, 0.238 mmol) in dry CH₂Cl₂ (1.0 mL) at 0° C. was added CF₃COOH(0.5 mL, 5 vol) and the reaction mixture was kept at this temperaturefor 30 min. It was allowed to come to rt and to stir at this temperaturefor 3 h. The reaction mixture was concentrated under reduced pressureand the residue was quenched with water (2 mL), was basified with NaHCO₃solution, and was extracted with ethyl acetate (2×8 mL). The combinedethyl acetate extract was washed with water (2 mL), brine (2 mL), driedover Na₂SO₄ and concentrated under reduced pressure to giveN⁴-(4-phenoxyphenyl)-N⁶-(piperidin-3-yl)pyrimidine-4,6-diamine (0.07 g,81%) as a light yellow solid.

Step 4

To a stirred solution ofN⁴-(4-phenoxyphenyl)-N⁶-(piperidin-3-yl)pyrimidine-4,6-diamine (0.025 g,0.069 mmol) in NMP (0.5 mL) at 0° C. was added acryloyl chloride (0.007g, 0.083 mmol), and the reaction mixture was stirred at 0° C. for 5 min.The reaction mixture was quenched by adding 10% NaHCO₃ solution and itwas extracted with EtOAc (2×5 mL). The combined EtOAc extract was washedwith water (3 mL) and brine (3 mL), was dried over Na₂SO₄ and wasconcentrated under reduced pressure. The residue obtained was purifiedby column chromatography (SiO₂, 230-400, chloroform/methanol:98/2) togive1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1one(I-86) as an off white solid. ¹H NMR (MeOD) 8 ppm: 1.5-1.75 (m, 2H),1.80-2.00 (m, 1H), 2.0-2.20 (m, 1H), 2.70-2.90 (m, 1H), 2.90-3.05 (m,1H), 3.80-4.00 (m, 2H), 4.30-4.45 (m, 1H), 5.65 & 5.75 (d, J=10.8 Hz &d, J=10.8 Hz respectively, together 1H), 5.81 (d, J=10.8 Hz, 1H), 6.14 &6.18 (d, J=17.2 Hz & d, J=19.2 Hz respectively, together 1H), 6.60-6.70& 6.70-6.85 (m, together 1H), 6.97-7.00 (m, 4H), 7.04 (t, J=7.6 Hz, 1H),7.32-7.38 (m, 4H), 8.04 & 8.07 (s, together 1H); LCMS: m/e 416.1 (M+1).

Example 27

Synthesis of3-methyl-1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)but-2-en-1-one(I-87)

Step 1

To a stirred solution of 4,6-dichloropyrimidine (0.5 g, 3.7 mmol) inn-butanol (10 mL) was added methyl 3-aminobenzoate (0.498 g, 3.7 mmol)and DIPEA (0.65 g, 5.0 mmol) and the reaction mixture was heated at 110°C. for 12 h. It was cooled and excess n-butanol was removed underreduced pressure. The residue was extracted with EtOAc (2×30 mL) and thecombined EtOAc extract was washed with water (5 mL), brine (2.5 mL),dried over Na₂SO₄ and concentrated under reduced pressure. The residuewas stirred with pet ether (30 mL) for 30 min, the pet ether removed bydecantation and the solid obtained was dried under high vacuum to givemethyl 3-(6-chloropyrimidin-4-ylamino)benzoate (0.3 g, 34%) as lightbrown solid.

Step 2

To a stirring solution of methyl 3-(6-chloropyrimidin-4-ylamino)benzoate(1.0 g, 3.8 mmol) and 4-phenoxy aniline (0.703 g, 3.8 mmol) in ethanol(5 mL) was added acetic acid (0.22 mL) and the reaction mixture washeated at 100° C. for 48 h. The reaction mixture was cooled, ethanol wasremoved under reduced pressure and the residue was dissolved in ethylacetate (50 mL). It was washed with NaHCO₃ solution (5 mL) and brine (5mL), was dried over Na₂SO₄ and was concentrated under reduced pressure.The residue was purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) to yield methyl3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzoate (1 g, 66%) as anoff-white solid.

Step 3

To a stirred solution of methyl3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzoate (0.3 g, 0.72mmol) in methanol/THF (2/2, 4 mL) was added LiOH (0.122 g, 2.9 mmol) inH₂O (4 mL) and the reaction mixture was stirred at rt for 2 h. It wasconcentrated under reduced pressure. the residue was diluted with water(2 mL) and was extracted with dichloromethane (5 mL). The aqueous layerwas separated and was acidified with 1.5N HCl (pH ˜5-6) to get a whiteprecipitate, which was collected by filtration and dried under vacuum togive 3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzoic acid (0.2 g69%) as a white solid.

Step 4

To a stirred solution of3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzoic acid (0.05 g,0.12 mmol) in DMF (2 mL) were added MeNH—OMe.HCl (0.0084 g, 0.12 mmol),EDCl.HCl (0.0361 g, 0.18 mmol), HOBT (0.0084 g, 0.062 mmol) and DIPEA(0.023 g, 0.18 mmol). The reaction mixture was stirred at roomtemperature for 1 h and was quenched with water. A white solid wasisolated by filtration and dried under vacuum to giveN-methoxy-N-methyl-3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzamide(0.025 g, 44.5%) as a white solid.

Step 5

To a stirred solution ofN-methoxy-N-methyl-3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzamide(50 mg, 0.11 mmol) in THF (0.5 mL) at 0° C. was added2-methylpropenylmagnesium bromide (1.1 mL, 0.55 mmol, 0.5 M in THF). Thereaction mixture was allowed to stir at room temperature for 30 min. Itwas quenched with sat. NH₄Cl solution (0.5 mL) and was extracted withEtOAc (3×2 mL). The combined organic layer was washed with brine, wasdried over anhydrous Na₂SO₄, was filtered and was concentrated underreduced pressure to give a white solid, which was further purified bycolumn chromatography (SiO₂, 60-120, chloroform/methanol, 9/1) to give3-methyl-1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)but-2-en-1-one(I-87) as an off white solid. ¹H NMR (CDCl₃) δ ppm: 2.01 (s, 3H), 2.20(s, 3H), 6.14 (s, 1H), 6.71 (s, 1H), 6.95 (s, 1H), 6.99-7.02 (m, 5H),7.11 (t, J=7.36 Hz, 1H), 7.26-7.28 (m, 2H), 7.34 (t, J=7.56 Hz, 2H),7.40-7.53 (m, 2H), 7.65 (d, J=7 Hz, 1H), 7.86 (s, 1H), 8.32 (s, 1H);LCMS: m/e 437.2 (M+1).

Example 28

Synthesis of1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)but-2-yn-1-one(I-88)

To a stirred solution ofN-methoxy-N-methyl-3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzamide(50 mg, 0.11 mmol) in THF (0.5 mL) at 0° C. was added a THF solution of1-butynylmagnesium bromide (1.1 mL, 1.1 mmol). The reaction mixture wasallowed to come to rt and was stirred at rt for 30 min. The reactionmixture was quenched with saturated NH₄Cl solution (0.5 mL) and wasextracted with EtOAc (2×3 mL). The combined EtOAc layer was washed withbrine, was dried over anhydrous Na₂SO₄, was filtered and wasconcentrated under reduced pressure to give a white solid, which h wasfurther purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) to give1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)but-2-yn-1-one(I-88) as an off-yellow solid. ¹H NMR (DMSO-d₆) 5 ppm: 2.22 (s, 3H),6.16 (s, 1H), 6.97 (d, J=8.6 Hz, 2H), 7.01 (d, J=8.8 Hz, 2H), 7.1 (t,J=7.2 Hz, 1H), 7.35-7.39 (m, 2H), 7.48 (t, J=8 Hz, 1H), 7.56 (d, J=8.8Hz, 2H), 7.66 (d, J=7.2 Hz, 1H), 7.95 (d, J=7.6 Hz, 1H), 8.32 (s, 1H),8.39 (s, 1H), 9.22 (s, 1H), 9.47 (s, 1H); LCMS: m/e 421.1 (M+1).

Example 29

Synthesis of(E,Z)-1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)but-2-en-1-one(I-89)

To a stirred solution ofN-methoxy-N-methyl-3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzamide(50 mg, 0.11 mmol) in THF (0.5 mL) at 0° C. was added a THF solution ofpropenylmagnesium bromide (2.2 mL, 1.1 mL, 0.5 M soln. in THF). Thereaction mixture was allowed to stir at room temperature for 30 min. Itwas quenched with saturated NH₄Cl solution (0.5 mL) and was extractedwith EtOAc (2×3 mL). The combined organic layer was washed with brine,was dried over anhydrous Na₂SO₄, was filtered and was concentrated underreduced pressure to get a white solid, which was further purified bycolumn chromatography (SiO₂, 60-120, chloroform/methanol, 9/1) to give(E,Z)-1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)but-2-en-1-one(I-89) as a pale yellow solid. ¹H NMR (DMSO-d₆) 8 ppm: 1.98 (dd, J=1.6 &6.8 Hz, 3H) & 2.13 (dd, J=1.6 & 7.2 Hz, 3H), 6.10-6.13 (m, 1H),6.75-6.90 (m, 1H), 6.90-7.13 (m, 7H), 7.25-7.27 (m, 1H), 7.32-7.34 (m,2H), 7.34-7.50 (m, 2H), 7.63-7.65 (m, 1H), 7.86-7.88 (m, 1H), 8.32 (s,1H); LCMS: m/e 423 (M+1).

Example 30

Synthesis of2-methyl-1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)prop-2-en-1-one(I-83)

To aN-methoxy-N-methyl-3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)benzamide(0.150 g, 0.340 mmol) at 0° C. was added 2-methylpropenylmagnesiumbromide (6.8 mL, 3.4 mmol, 0.5 M solution in THF). The reaction mixturewas allowed to stir at room temperature for min. It was quenched withsaturated NH₄Cl solution (0.5 mL) and was extracted with EtOAc (2×3 mL).The combined organic layer was washed with brine, dried over Na₂SO₄,filtered and concentrated under reduced pressure to get a white solid,which was further purified by column chromatography (SiO₂, 60-120,product getting eluted in methanol/chloroform:2/98) to give2-methyl-1-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)prop-2-en-1-one(I-83) as a white solid. ¹H NMR (DMSO-d₆) δ ppm: 1.99 (s, 3H), 5.64 (s,1H), 6.03 (s, 1H), 6.14 (s, 1H), 6.96-7.02 (m, 4H), 7.10 (t, J=7.6 Hz,1H), 7.25 (d, J=7.6 Hz, 1H), 7.35-7.43 (m, 3H), 7.56 (d, J=8.8 Hz, 2H),7.88 (d, J=8 Hz, 1H), 7.92 (s, 1H), 8.29 (s, 1H), 9.21 (s, 1H), 9.38 (s,1H); LCMS: 423 m/e (M+1).

Example 31

Synthesis ofN-(6-(6-(3-methoxyphenoxy)pyrimidin-4-ylamino)pyridine-2-yl)acrylamide(I-90)

Step 1

To a solution of N²-(6-chloropyrimidin-4-yl)pyridine-2,6-diamine (200mg, 0.90 mmol) in dry DMF (2 mL) was added 3-methoxyphenol (112 mg, 0.90mmol) and anhydrous K₂CO₃ (186 mg, 1.353 mmol). The reaction mixture washeated at 100° C. for 16 h under N₂ atmosphere. It was then cooled andDMF removed under reduced pressure to give a yellowish gummy residuethat was taken in EtOAc (10 mL). It was washed with water (5 mL), brine(5 mL), dried over Na₂SO₄ and then concentrated under reduced pressureto give a crude product. This was further purified by columnchromatography (SiO₂, 60-120, hexane/EtOAc, 5/5) to giveN²-(6-(3-methoxylphenoxy)pyrimidin-4-yl)pyridine-2,6-diamine (110 mg,40.7%) as a pale yellow solid.

Step 2

To a stirred solution ofN²-(6-(3-methoxylphenoxy)pyrimidin-4-yl)pyridine-2,6-diamine (100 mg,0.323 mmol) in THF/NMP (1 mL/0.5 mL) was added acryloyl chloride (0.029g, 0.3 mmol) under N₂ atmosphere at −10° C. The stirring was continuedat the same temperature for 2 h and the reaction mixture wasconcentrated under reduced pressure to give a residue that was furtherpurified by column chromatography (SiO₂, 60-120, chloroform/methanol) togiveN-(6-(6-(3-methoxyphenoxy)pyrimidin-4-ylamino)pyridine-2-yl)acrylamide(I-90) as a pale yellow solid. ¹H NMR (DMSO-d₆) ppm: 3.75 (s, 3H), 5.79(dd, J=1.8 & 10.08 Hz, 1H), 6.30 (dd, J=1.8 & 16.96 Hz, 1H), 6.65 (dd,J=10.12 & 16.96 Hz, 1H), 6.74-6.76 (m, 2H), 6.82 (td, J=1.52 & 9.24 Hz,1H), 7.04 (d, J=7.92 Hz, 1H), 7.33 (t, J=8.28 Hz, 1H), 7.70 (t, J=7.96Hz, 1H), 7.76 (d, J=8 Hz, 1H), 7.90 (s, 1H), 8.34 (s, 1H), 10.20 (s,1H), 10.48 (s, 1H); LCMS: m/e 364 (M+1).

Example 32

Synthesis ofN-(6-(6-(4-methoxyphenoxy)pyrimidin-4-ylamino)pyridine-2-yl)acrylamide(I-91)

Step 1

To a solution of N²-(6-chloropyrimidin-4-yl)pyridine-2,6-diamine (200mg, 0.90 mmol) in dry DMF (2 mL) was added 4-methoxyphenol (168 mg, 1.3mmol) and anhydrous K₂CO₃ (179 mg, 1.3 mmol). The reaction mixture washeated at 100° C. for 16 h under N₂ atmosphere. It was then cooled andDMF removed under reduced pressure to give a yellowish gummy residuethat was taken in EtOAc (10 mL). The solution was washed with water (5mL) and brine (5 mL), was dried over Na₂SO₄ and was then concentratedunder reduced pressure to get crude product. This was further purifiedby column chromatography (SiO₂, 60-120, hexane/EtOAc, 5/5) to giveN²-(6-(4-methoxylphenoxy)pyrimidin-4-yl)pyridine-2,6-diamine (160 mg,59.2%) as a pale yellow solid.

Step 2

To a stirred solution ofN²-(6-(4-methoxylphenoxy)pyrimidin-4-yl)pyridine-2,6-diamine (150 mg,0.474 mmol) in THF/NMP (1 mL/0.5 mL) was added acryloyl chloride (64 mg,0.712 mmol) at −10° C. under N₂ atmosphere. After stirring at thistemperature for 30 min, the reaction was stopped and the reactionmixture was slowly added to NaHCO₃ solution (10 mL). A white solid wasprecipitated which was isolated by filtration and was dissolved in amixture of ethyl acetate (5 mL) and Et₃N (0.5 mL). The solution waswashed with water (2 mL) and brine (2 mL). Drying over Na₂SO₄ followedby filtration and concentration under reduced pressure afforded a yellowsolid. It was further purified by column chromatography (SiO₂, 60-120,chloroform/methanol, 9/1) to giveN-(6-(6-(4-methoxyphenoxy)pyrimidin-4-ylamino)pyridine-2-yl)acrylamide(I-91) as an off white solid. ¹H NMR (DMSO-d₆) δ ppm: 3.76 (s, 3H), 5.79(dd, J=1.84 & 10.16 Hz, 1H), 6.30 (dd, J=1.84 & 17 Hz, 1H), 6.65 (dd,J=10.12 & 16.92 Hz, 1H), 6.96-6.99 (m, 2H), 7.02 (d, J=7.88 Hz, 1H),7.09-7.13 (m, 2H), 7.69 (t, J=7.96 Hz, 1H), 7.76 (d, J=7.44 Hz, 1H),7.86 (s, 1H), 8.30 (d, J=0.88 Hz, 1H), 10.17 (s, 1H), 10.17 (s, 1H);LCMS: m/e 364 (M+1).

Example 33

Synthesis ofN-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)propionamide(I^(R)-10)

Step 1

A solution of tert-butyl 3-(6-chloropyrimidin-4-ylamino)phenylcarbamate(200 mg, 0.6 mmol), 4-phenoxyaniline (346 mg, 1.8 mmol) and concentratedHCl (45 mg, 1.2 mmol) in n-butanol (8 mL) was subjected to microwaveirradiation (160° C., 20 min). The reaction mixture was quenched withNaHCO₃ solution (2 mL) and extracted with EtOAc (2×10 mL). The combinedEtOAc extract was washed with water (5 mL), brine (5 mL), was dried overNa₂SO₄ and was concentrated under reduced pressure. The residue wasfurther purified by column chromatography (SiO₂, 60-120,Chloroform/methanol, 9/1) to giveN⁴-(3-aminophenyl)-N⁶-(4-phenoxyphenyl)pyrimidine-4,6-diamine (87 mg,37.8%) as a brown solid.

Step 2

To a solution of propionic acid (12 mg, 0.1 mmol) in DMF (0.6 mL) wasadded HATU (92 mg, 0.2 mmol) and the reaction mixture was stirred atroom temperature for 30 min. To it was addedN⁴-(3-aminophenyl)-N⁶-(4-phenoxyphenyl)pyrimidine-4,6-diamine (60 mg,0.1 mmol) followed by DIPEA (41 mg, 0.3 mmol) and the reaction mixturewas stirred at this temperature for 16 h. It was concentrated underreduced pressure. The residue was diluted with CH₂Cl₂ (5 mL) and waswashed with NaHCO₃ solution (2 mL), water (2 mL), and brine (2 mL).Drying over Na₂SO₄ followed by concentration under reduced pressureoffered a residue that was purified by column chromatography (SiO₂,230-400, chloroform/methanol:9/1) to giveN-(3-(6-(4-phenoxyphenylamino)pyrimidin-4-ylamino)phenyl)propionamide(I^(R)-10) as a brown solid. ¹H NMR (DMSO-d₆) δ ppm: 1.06 (t, J=7.56 Hz,3H), 2.30 (q, J=7.48 Hz, 2H), 6.13 (s, 1H), 6.94-6.99 (m, 4H), 7.08 (t,J=7.36 Hz, 1H), 7.16-7.24 (m, 3H), 7.36 (t, J=7.44 Hz, 2H), 7.54 (d,J=8.88 Hz, 2H), 7.81 (s, 1H), 8.23 (s, 1H), 9.12 (s, 2H), 9.81 (s, 1H);LCMS: m/e 426.3 (M+1).

Example 34

Synthesis of(E)-N-(3-(6-(3-chloro-4-fluorophenoxy)pyrimidin-4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide(I-92)

Step 1

A solution of tert-butyl 3-(6-chloropyrimidin-4-ylamino)phenylcarbamate(1.6 g, 5 mmol), 3-chloro-4-fluorophenol (1.4 g, 10 mmol) and potassiumcarbonate (1.4 g, 10 mmol) in 15 mL of DMF was heated to 120° C. for 16h. The reaction mixture was mixed in 15 mL of water, the crude productwas precipitated, filtered, purified by flash chromatography on silicagel with MeOH/DCM solvent system to afford 750 mg (45% yield) ofN¹-(6-(3-chloro-4-fluorophenoxy)pyrimidin-4-yl)benzene-1,3-diamine as anoff-white solid. MS (m/z): MH⁺=331.

Step 2

Oxalyl chloride (155 mg, 1.2 mmol) was added dropwise to a mixture of4-N,N-dimethyl aminocrotonic acid HCl salt (200 mg, 1.2 mmol) in 5 mL ofTHF at 0° C. To this mixture was added 3 drops of DMF/THF solution (madefrom 5 drops of DMF in 1 mL of THF). The reaction mixture was stirred atRT for 2 h, then was cooled to 0° C. ice bath. A solution ofN′-(6-(3-chloro-4-fluorophenoxy)pyrimidin-4-yl)benzene-1,3-diamine (200mg, 0.6 mmol) 2 mL of NMP) was added to the dimethylaminocrotonylchloride solution and the resulting mixture was stirred for 3 h at 0° C.The reaction was quenched with 3 mL of 1N NaOH, and was extracted withEtOAc (2×25 mL). The crude product mixture was purified by flashchromatography on silica gel with MeOH/NH₄OH/DCM solvent system toafford(E)-N-(3-(6-(3-chloro-4-fluorophenoxy)pyrimidin-4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide(I-92) as a light colored solid. MS (m/z): MH⁺=442, 444(3:1), ¹H-NMR(DMSO) δ 10.08 (s, 1H), 9.67 (s, 1H), 8.36 (s, 1H), 7.98 (s, 1H), 7.59(d, 1H), 7.57 (t, 1H), 7.53-7.24 (m, 4H), 6.29 (b, 1H), 6.23 (s, 1H),3.51 (d, 2H), 2.21 (s, 6H).

Example 35

Synthesis of1-(3-(6-(3-chloro-4-fluorophenyamino)pyrimidin-4-ylamino)pyrrolidin-1-yl)prop-2-en-1-one(I-70)

Step 1

A neat mixture of tert-butyl3-(6-chloropyrimidin-4-ylamino)pyrrolidin-1-carboxylate (354 mg, 1.18mmol) and 3-chloro-4-fluoroaniline (3.03 g, 20.8 mmol) was heated at140° C. for 21 hours. Upon cooling to ambient temperature, the melt wasdiluted with EtOAc and the mixture was stirred for 1 hour. A beige,amorphous precipitate was collected, washed with water and dried invacuo at 50-60° C. giving 292 mg (80%) ofN⁴-(3-chloro-4-fluorophenyl)-N⁶-(pyrrolidin-3-yl)pyrimidine-4,6-diamine.MS (APCI): (M+1)=308, (M−1)=306.

Step 2

To a solution ofN⁴-(3-chloro-4-fluorophenyl)-N⁶-(pyrrolidin-3-yl)pyrimidine-4,6-diamine(287 mg, 0.93 mmol) and triethylamine (0.32 ml, 2.33 mmol) in anhydrousTHF (5 ml) under nitrogen was added acryloyl chloride (91 μl, 1.12mmol). The reaction mixture was stirred at room temperature for 1 hourand was concentrated under reduced pressure. The residue was elutedthrough a flash column (silica gel 60, 230-400 mesh, 5% MeOH in EtOAc to10% MeOH in EtOAc) to give two products. The less polar product(R_(f)=0.24 in 1:9 MeOH:EtOAc) was found to be a diacrylated analog. Themore polar product (R_(f)=0.14 in 1:9 MeOH:EtOAc) was1-(3-(6-(3-chloro-4-fluorophenyamino)pyrimidin-4-ylamino)pyrrolidin-1-yl)prop-2-en-1-one(I-70). MS (APCI) (M+1)=362, (M−1)⁺=360: ¹H-NMR DMSO-d₆) δ 9.11 (s, 1H),8.14 (s, 1H), 7.92 (d, 1H), 7.38-7.23 (m, 3H), 6.58-6.48 (m, 1H),6.13-6.08 (m, 1H), 5.75 (d, 1H) 5.63-5.59 (m, 1H), 3.64-3.59 (m, 2H),2.17-1.81 (m, 6H).

Example 36

Synthesis of1-(3-(6-(3-chloro-4-fluorophenyamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-one(I-71)

Step 1

N-(3-Chloro-4-fluorophenyl)-N′-piperidin-3-yl pyrimidine-4,6-diamine.

A mixture of tert-butyl3-(6-chloropyrimidin-4-ylamino)piperidine-1-carboxylate (295 mg, 0.94mmol) and 3-chloro-4-fluoroaniline (2.83 g, 19.4 mmol) was heated neatat 140° C. for 19 hours. Upon cooling, the melt was stirred in EtOAc andallowed to stand at room temperature for 1 hour. The precipitate wascollected, washed with EtOAc and dried to give 256 mg (85%) ofN⁴-(3-chloro-4-fluorophenyl)-N⁶-(piperidin-3-yl)pyrimidine-4,6-diamine,a grayish-violet amorphous solid. MS (APCI): (M+1)⁺=322.

Step 2

To a solution ofN⁴-(3-chloro-4-fluorophenyl)-N⁶-(piperidin-3-yl)pyrimidine-4,6-diamine(251 mg, 0.78 mmol) and triethylamine (0.27 ml, 1.95 mmol) in anhydrousTHF (7 ml) under nitrogen was added acryloyl chloride (76 μl, 0.94mmol). The reaction mixture was stirred at room temperature for 1 hourand was concentrated under reduced pressure. The residue was elutedthrough a flash column (silica gel 60, 230-400 mesh with 5% MeOH inEtOAc to give two products. The less polar product (R_(f)=0.33 in 1:9MeOH:EtOAc) was found to be a diacrylated analog. The more polar product(R_(f)=0.24 in 1:9 MeOH:EtOAc) was1-(3-(6-(3-chloro-4-fluorophenyamino)pyrimidin-4-ylamino)piperidin-1-yl)prop-2-en-1-one(I-71). MS (APCI) (M+1)⁺=376, (M−1)⁺=374: ¹H-NMR DMSO-d₆, δ 9.15 (br s,1H), 8.18 (d, 1H), 7.95 (br s, 1H), 7.43-7.20 (m, 2H), 7.03-6.48 (m,3H), 6.26-5.97 (m, 2H), 5.86-5.51 (m, 3H), 3.90-3.66 (m, 2H), 1.98-1.66(m, 2H), 1.59-1.12 (m, 2H).

Example 37

Synthesis ofN-(6-(6-(2,3-dihydrobenzo[b][1,4]dioxin-6-yloxy)pyrimidin-4-ylamino)pyridin-2-yl)acrylamide(I-95)

Step 1

To a stirred solution of 2,3-dihydrobenzo[b][1,4]dioxine-6-carbaldehyde(1 g, 6.09 mmol) in CH₂Cl₂ (16 mL) was added m-CPBA (4.204 g, 24.36mmol). The suspension was heated at 50° C. for 2 days, was cooled to rt,was quenched with saturated NaHCO₃ soln. and was extracted with CH₂Cl₂(3×10 mL). The combined extract was concentrated under reduced pressure,and then was dissolved in MeOH containing NaOH. The solution was stirredat rt for 2 h, was acidified with HCl and was extracted with ethylacetate (3×10 mL). The combined extract was washed with saturated NaHCO₃solution and brine, was dried over anhydrous Na₂SO₄, was filtered andwas concentrated under reduced pressure. The residue was dissolved inCH₂Cl₂ and was filtered. The DCM solution was dried over anhydrousNa₂SO₄, was filtered and was concentrated under reduced pressure to give2,3-dihydrobenzo[b][1,4]dioxin-6-ol (0.591 g, 63%) as a reddish brownoily liquid.

Step 2

A stirred mixture of 2,3-dihydrobenzo[b][1,4]dioxin-6-ol (0.137 g, 0.90mmol), Cs₂CO₃ (0.734 g, 2.25 mmol), CuI (0.02 g, 10% w/w), andN²-(6-chloropyrimidin-4-yl)pyridine-2,6-diamine (0.29 g, 0.90 mmol) inNMP (1 mL) was heated at 100° C. for 16 h. The reaction mixture wascooled to rt and slowly added to demineralised water. The solid thatprecipitated was collected by filtration and was further purified bycolumn chromatography (SiO₂, 60-120, pet ether/ethyl acetate, 7/3) togiveN²-(6-(2,3=dihydrobenzo[b][1,4]dioxin-6-yloxy)pyrimidin-4-yl)pyridine-2,6-diamine(0.088 g, 29%) as yellow solid.

Step 3

To a stirred solution ofN²-(6-(2,3-dihydrobenzo[b][1,4]dioxin-6-yloxy)pyrimidin-4-yl)pyridine-2,6-diamine(0.080 g, 0.23 mmol) and potassium carbonate (0.065 g, 0.47 mmol) in NMP(0.8 mL) at 0° C. was added acryloyl chloride (0.026 g, 0.29 mmol) andthe reaction mixture was stirred at 0° C. for 30 min. The reactionmixture was added dropwise to a cold, stirring solution of 10% NaHCO₃and was stirred at 0° C. for 30 min. A white solid was isolated byfiltration. The solid was washed with cold water and hexane anddissolved in a 2 mL of methanol/dichloromethane (1/1). This solution wasconcentrated under reduced pressure. The residue obtained was suspendedin cold water (5 mL), Et₃N was added to it and it was extracted withethyl acetate (2×5 mL). The combined ethyl acetate extract was washedwith water (2 mL) and brine (2 mL), was dried over Na₂SO₄ and wasconcentrated under reduced pressure to obtainN-(6-(6-(2,3-dihydrobenzo[b][1,4]dioxin-6-yloxy)pyrimidin-4-ylamino)pyridin-2-yl)acrylamide(I-95) as a light yellow solid. ¹H NMR (DMSO-d₆) δ ppm: 4.25 (s, 4H),5.79 (dd, J=1.84 & 10.08 Hz, 1H), 6.30 (dd, J=1.84 & 16.96 Hz, 1H),6.62-6.72 (m, 3H), 6.88 (d, J=8.72 Hz, 1H), 7.03 (d, J=7.84 Hz, 1H),7.69 (t, J=7.96 Hz, 1H), 7.70 (d, J=7.84 Hz, 1H), 7.85 (s, 1H), 8.32 (d,J=0.4 Hz, 1H), 10.16 (s, 1H), 10.47 (s, 1H); LCMS: m/e 392.

Example 38 Synthesis ofN-(3-(6-(4-phenoxyphenoxy)pyrimidin-4-ylamino)phenyl)acrylamide (I-97)

The title compound was prepared according to the schemes, steps andintermediates described below.

Step 1

To a stirred solution of 2 (4.35 g, 23.38 mmol) and Cs₂CO₃ (15.26 g,46.76 mmol) in dry DMF (50 mL) was added 1 (5.0 g, 15.58 mmol) at rt andthe reaction was stirred at 80° C. for 16 h under nitrogen atmosphere.The reaction mixture was cooled, concentrated under reduced pressure andthe residue was diluted with ethyl acetate (50 mL). It was washed withwater (2×10 mL), brine (10 mL), dried over Na₂SO₄ and concentrated underreduced pressure. The residue was further purified by columnchromatography (SiO₂, 60-120, pet ether/ethyl acetate, 5/5) to give 3(1.6 g, 23.3%) as brown solid.

Step 2

To a stirred solution of 3 (1.6 g, 3.9 mmol) in dry CH₂Cl₂ (8 mL) at 0°C. was added CF₃COOH (4.8 mL) and the reaction mixture was stirred at 0°C. for 30 min. The reaction was allowed to come to rt and stirred for 12h. It was concentrated under reduced pressure and the residue wasquenched with NaHCO₃ solution (15 mL). The contents were extracted withethyl acetate (3×15 mL) and the combined extracts were washed with water(5 mL), brine (5 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give 4 (1.2 g, 99%) as a light yellow solid.

Step 3

To a stirred solution of 4 (1.2 g, 3.23 mmol) and potassium carbonate(2.237 g, 16.19 mmol) in NMP (12 mL) at 0° C. was added acryloylchloride (0.322 g, 3.56 mmol) and the reaction mixture was stirred at 0°C. for 60 min The reaction mixture was added dropwise to a cold,stirring solution of 10% NaHCO₃ and stirred at the same temperature (0°C.) for 30 min. A solid precipitated out and was isolated by filtrationthrough a Buchner funnel. It was washed with cold water and hexane,dissolved in a mixture of methanol/dichloromethane (50:50, 5 mL), andconcentrated under reduced pressure. The residue obtained was suspendedin cold water (10 mL), Et₃N was added to it, and it was extracted withethyl acetate (2×10 mL). The combined ethyl acetate extract was washedwith water (5 mL), brine (5 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The residue was further purified by columnchromatography (SiO₂, 60-120, methanol/chloroform: 10/90) to give thetitle compound (0.83 g, 60.3%) as a light green solid. ¹H NMR (DMSO-d₆)δ ppm: 5.74 (dd, J=1.92 & 10.04 Hz, 1H), 6.16 (s, 1H), 6.24 (dd, J=1.92& 16.92 Hz, 1H), 6.45 (dd, J=10.08 & 16.96 Hz, 1H), 7.03-7.09 (m, 4H),7.16 (t, J=7.30 Hz, 1H), 7.20-7.26 (m, 3H), 7.30-7.35 (m, 2H), 7.39-7.44(m, 2H), 7.98 (s, 1H), 8.36 (s, 1H), 9.62 (s, 1H), 10.15 (s, 1H); LCMS:m/e 425.2 (M+1).

Example 39 Synthesis ofN-(3-(morpholine-4-carbonyl)-5-(6-(4-phenoxyphenoxy)pyrimidin-4-ylamino)phenyl)acrylamide(I-100)

The title compound was prepared according to the schemes, steps andintermediates described below.

Step 1

To a stirring solution of 1 (2.0 g, 10.75 mmol) and K₂CO₃ (2.96 g, 21.5mmol) in dry DMF (20 mL) was added 2 (1.59 g, 10.75 mmol) and thereaction mixture was stirred at rt for 16 h under nitrogen atmosphere.It was cooled, quenched with water (100 mL) and extracted with EtOAc(2×50 mL). The combined EtOAc extract was washed with 10% NaHCO₃ soln.(50 mL), water (3×50 mL), brine (50 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to give 3 (1.92 g, 60.0%) as a lightyellow solid, which was used in the next step without furtherpurifications.

Step 2

To a solution of 3 (0.5 g, 1.678 mmol) in ethanol (10 mL) was addedconc. HCl (0.172 g, 1.678 mol) and 4 (1.27 g, 8.34 mol). The reactionmixture was stirred in a sealed pressure tube for 10 h at 90° C. Thereaction was cooled, quenched with water (50 mL) and extracted withEtOAc (2×25 mL). The combined extracts were washed with 5% citric acidsolution (25 mL), water (25 mL), brine (25 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to get 5 (0.4 g, 57.53%) as a brownsolid.

Step 3

To a stirred solution of 5 (0.15 g, 0.362 mmol) in DMF (6 mL) were addedmorpholine (0.095 g, 1.085 mmol), EDCI.HCl (0.104 g, 0.542 mmol), HOBT(0.0049 g, 0.036 mmol) and DIPEA (0.07 g, 0.543 mmol). The reactionmixture was stirred at room temperature for 18 h under nitrogenatmosphere. It was diluted with cold water (30.0 mL) and extracted withEtOAc (2×25 mL). The combined extracts were washed with water (2×15 mL),brine (10 mL), dried over Na₂SO₄ and concentrated under reduced pressureto give crude 6. The crude residue was further purified by columnchromatography (SiO₂. MeOH/Chloroform:2/98) to get 6 (0.075 g, 42.87%)as a brown solid.

Step 4

To a stirred solution of 6 (0.09 g, 0.186 mmol) and potassium carbonate(0.128 g, 0.93 mmol) in NMP (1.5 mL) at 0° C. was added 7 (0.021 g,0.232 mmol) and the reaction mixture was stirred at 0° C. for 30 min Thereaction mixture was added drop wise to a cold, stirring solution of 10%NaHCO₃ and stirred at the same temperature (0° C.) for 5 in. A solidprecipitated out which was isolated by filtration through a Buchnerfunnel. The solid was washed with cold water, hexane and dissolved in amixture of methanol/dichloromethane (50:50, 5 mL) and concentrated underreduced pressure. The residue obtained was suspended in cold water (5mL), Et₃N was added, and it was extracted with ethyl acetate (2×15 mL).The combined extracts were washed with water (10 mL), brine (10 mL),dried over Na₂SO₄ and concentrated under reduced pressure to give thetitle compound (0.08 g, 79.95%) as a pale brown solid. ¹H NMR (DMSO-d₆)δ ppm: 3.62 (bs, 8H), 5.79 (dd, J=1.6 & 10.4 Hz, 1H), 6.18 (s, 1H), 6.28(dd, J=1.6 & 16.8 Hz, 1H), 6.41-6.50 (m, 1H), 7.05-7.10 (m, 4H), 7.17(t, J=7.2 Hz, 1H), 7.24 (d, J=8.8 Hz, 2H), 7.39 (s, 1H), 7.43 (t, J=8.4Hz, 2H), 7.51 (s, 1H), 8.0 (s, 1H), 8.41 (s, 1H), 9.79 (s, 1H), 10.31(s, 1H); LCMS: m/e 538.2 (M+1).

Example 40 Synthesis of3-acrylamido-N-(2-methoxyethyl)-5-(6-(4-phenoxyphenoxy)pyrimidin-4-ylamino)benzamide(I-102)

The title compound was prepared according to the schemes, steps andintermediates described in Example 39, by using 2-methoxyethylamine instep-3. ¹H NMR (DMSO-d₆) δ ppm: 3.25 (s, 3H), 3.30-3.50 (m, 4H), 5.76(dd, J=1.84 & 10.08 Hz, 1H), 6.17 (s, 1H), 6.27 (dd, J=1.88 & 16.96 Hz,1H), 6.46 (dd, J=10.12 & 16.92 Hz, 1H), 7.03-7.09 (m, 4H), 7.16 (dt,J=0.88 & 7.4 Hz, 1H), 7.22 (td, J=3.28 & 9.96 Hz, 2H), 7.41 (dt, J=1.92& 7.6 Hz, 2H), 7.70 (d, J=1.64 Hz, 2H), 8.17 (s, 1H), 8.38 (s, 1H), 8.42(t, J=5.28 Hz, 1H), 9.75 (S, 1H), 10.30 (s, 1H); LCMS: m/e 526.2 (M+1).

Example 41 Synthesis ofN-(3-(6-(4-phenoxyphenylthio)pyrimidin-4-ylamino)phenyl)acrylamide(I-103)

The title compound was prepared according to the schemes, steps andintermediates described below.

Step 1

To a stirring solution of 2 (0.25 g, 1.235 mmol) and K₂CO₃ (0.512 g,3.705 mmol) in dry DMF (2.5 mL) was added 1 (0.320 g, 1.235 mmol) at rt,and the reaction was stirred at 110° C. for 16 h under nitrogenatmosphere. The reaction mixture was quenched with water and extractedwith ethyl acetate (3×5 mL). The combined extracts were washed withwater (2×25 mL), brine (25 mL), dried over Na₂SO₄ and concentrated underreduced pressure to give 3 (0.130 g, 21%) as a white solid, which wasused in the next step without purification.

Step 2

To a stirred solution of 3 (0.170 g, 0.349 mmol) in dry CH₂Cl₂ (2 mL) at0° C. was added CF₃COOH (1 mL) and the reaction mixture was stirred at0° C. for 30 min. The reaction was allowed to come to rt and stirred atit for 2 h. The reaction mixture was concentrated under reduced pressureand the residue was quenched with NaHCO₃ solution (8 mL). The contentswere extracted with ethyl acetate (3×5 mL) and the combined extractswere washed with water (5 mL), brine (5 mL), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, 60-120, product gettingeluted in 3% methanol/chloroform) to give 4 (0.100 g, 74%).

Step 3

To a stirred solution of 6 (0.095 g, 0.24 mmol) and potassium carbonate(0.169 g, 1.22 mmol) in NMP (0.95 mL) at 0° C. was added 7 (0.024 g,0.27 mmol), and the reaction mixture was stirred at 0° C. for 30 min.The reaction mixture was added dropwise to a cold, stirring solution of10% NaHCO₃ and stirred at the same temperature (0° C.) for 5 min. Asolid precipitated out and was isolated by filtration through a Buchnerfunnel. The solid was washed with cold water and hexane, dissolved in amixture of methanol/dichloromethane (50:50, 5 mL), and concentratedunder reduced pressure. The residue obtained was suspended in cold water(5 mL), Et₃N was added, and it was extracted with ethyl acetate (2×15mL). The combined ethyl acetate extract was washed with water (10 mL),brine (10 mL), dried over Na₂SO₄ and concentrated under reduced pressureto get further to give the title compound (61 mg, 56%) as a yellowsolid. ¹H NMR (DMSO-d₆) δ ppm: 5.75 (dd, J=1.96 & 10.04 Hz, 1H), 6.19(d, J=0.84 Hz, 1H), 6.25 (dd, J=2 & 16.92 Hz, 1H), 6.45 (dd, J=10.08 &16.96 Hz, 1H), 7.11-7.15 (m, 4H), 7.20-7.30 (m, 4H), 7.48 (dt, J=1.88 &6.56 Hz, 2H), 7.65 (dd, J=2.04 & 6.64 Hz, 2H), 7.97 (s, 1H), 8.41 (d,J=0.56 Hz, 1H), 9.55 (s, 1H), 10.15 (s, 1H); LCMS: m/e 441 (M+1).

Example 42 Synthesis ofN-(2-morpholino-5-(6-(4-phenoxyphenoxy)pyrimidin-4-ylamino)phenyl)acrylamide(I-101)

The title compound was prepared according to the schemes, steps andintermediates described below.

Step-1

4-Fluoro-3-nitroaniline (1, 1 g, 1 equiv.) and morpholine (1.67 g, 3equiv.) were dissolved in THF (10 mL). The reaction mixture was heatedat reflux overnight. After cooling, water/brine (10 mL) was added, themixture was agitated, and the layers were separated. The organic phasewas dried over sodium sulfate, and the solvent was removed via rotaryevaporation. The material was purified by flash chromatography using0-40% gradient of heptane/EtOAc to give 800 mg of 3 as an orange-redsolid.

Step-2

4-Chloro-6-(4-phenoxyphenoxy)pyrimidine (4, 200 mg, 1 equiv.) and3-nitro-4-morpholinoaniline (164 mg, 1.1 equiv.) were dissolved indioxane (5 mL). The solution was degassed for 1 min. Palladium acetate(22 mg, 5 mol %), X-Phos ligand (39 mg, 10 mol %) and CsCO₃ (436 mg, 2equiv.) were added in that order. The suspension was degassed for 1 minand placed under an argon atmosphere, and the mixture was heated toreflux for 12 h. After cooling, solvent was removed via rotaryevaporation. The dark oil was partitioned between water/brine and EtOAc(5 mL each), agitated, filtered off precipitate, and separated layers ofthe filtrate. The organic phase was dried over sodium sulfate. Thesolvent was removed via rotary evaporation to give a dark oil. The oilwas purified by flash chromatography using 0-30% gradient ofheptane/EtOAc to afford 100 mg of 5 as an orange-red solid.

Step-3

A catalytic amount of 10% Pd/C was added to a solution of 5 (100 mg) inEtOH (3 mL). Using a balloon, hydrogen was introduced, and the reactionwas stirred at rt for 12 h. The reaction mixture was filtered throughCelite, and the filtrate was concentrated via rotary evaporation to give6 as a dark purple film.

Step-4

A solution of 6 (100 mg, 1 equiv.) in THF (4 mL) was cooled inwater/ice-MeOH bath (−10° C. Acryloyl chloride was added to the solution(18.6 μL, 1.05 equiv.) and stirred for min, then Hunig's base was added(31.7 μL, 1.05 equiv.) and stirred for 10 min. Water/brine (5 mL) wasadded, agitated and the layers were separated. The organic phase wasdried over sodium sulfate. The solvent was removed via rotaryevaporation afford a dark oil. The oil was purified by flashchromatography using 30-80% heptane/EtOAc gradient to give the titlecompound as a red film. LC/MS (RT=3.029/(M+H)) 510.2

Example 43 Synthesis of1-(6-(6-(3-chlorophenoxy)pyrimidin-4-ylamino)-2,2-dimethyl-2H-benzo[b][1,4]oxazin-4(3H)-yl)prop-2-en-1-one(I-104)

The title compound was prepared according to the schemes, steps andintermediates described in Example 42, by omitting step-1, using4-tert-butylcarboxy-6-amino-2,2-dimethyl-2H-benzo[b][1,4]oxazine instep-2 and using TFA in CH₂Cl₂ in lieu of hydrogenation in step-3. LC/MS(R_(T)=3.035/(M+H)) 437.1

Example 44 Synthesis ofN-(2-morpholino-5-(6-(3-chlorophenoxy)pyrimidin-4-ylamino)phenyl)acrylamide(I-105)

The title compound was prepared according to the schemes, steps andintermediates described in Example 42 by using4-chloro-6-(3-chlorophenoxy)pyrimidine in place of 4 in step-2. LC/MS(R_(T)=2.957/(M+H)) 452.1.

Example 45 Synthesis ofN¹-(4-(((E)-4-(3-(6-(3-chloro-4-fluorophenylamino)pyrimidin-4-ylamino)-4-methoxyphenylamino)-4-oxobut-2-enyl)(methyl)amino)butyl)-Ns-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide(I-99)

The title compound was prepared according to the schemes, steps, andintermediates described below.

Step-1

The preparation ofN⁴-(5-amino-2-methoxyphenyl)-N⁶-(3-chloro-4-fluorophenyl)pyrimidine-4,6-diamine(1) is described in Example 7 (Int-G). To a stirred solution of 1 (150mg, 0.41 mmol) in DMF (8 mL) was added 2 (137 mg, 0.83 mmol), HATU (317mg, 0.83 mmol) and DIPEA (215 mg, 1.67 mmol) at 0° C. The reactionmixture was stirred for 18 h at RT. After the completion of reaction(monitored by TLC), the crude mixture was diluted with water andextracted with EtOAc (4×100 mL). The organic layers were dried overanhydrous Na₂SO₄ and concentrated under vacuo to afford 3 (100 mg,47.3%) as off-white solid, which was used in the next step withoutfurther purification. ¹H NMR (CDCl₃, 500 MHz): δ 8.35 (s, 1H), 8.12 (s,1H), 7.48-7.43 (m, 1H), 7.24 (s, 1H), 7.16-7.10 (m, 2H), 7.03 (s, 1H),6.84 (t, J=9.0 Hz, 2H), 6.49 (s, 1H), 6.15 (s, 1H), 5.29 (s, 1H), 3.85(s, 3H), 3.33 (d, J=5.0 Hz, 2H), 2.80 (s, 3H). Mass: m/z 506 (M⁺+1).

Step-2

To a stirred solution of 3 (300 mg, 0.59 mmol) and 4 (179 mg, 0.89 mmol)in acetone (25 mL) was added K₂CO₃ (163 mg, 1.88 mmol) at RT, and thereaction mixture was stirred for 18 h at RT. After the completion ofreaction (monitored by TLC), the reaction mixture was diluted with water(200 mL) and extracted with EtOAc (7×100 mL). The organic layers werewashed with water (4×100 mL), dried over anhydrous Na₂SO₄ andconcentrated under vacuo. The crude compound was purified by columnchromatography to afford 5 (205 mg, 55.1%) as off-white solid. ¹H NMR(DMSO-d₆, 500 MHz): δ 9.91 (s, 1H), 9.24 (s, 1H), 8.41 (s, 1H), 8.23 (s,1H), 7.95 (dd, J=2.5, 6.5 Hz, 1H), 7.90 (s, 1H), 7.47 (dd, J=2.5, 9.0Hz, 1H), 7.47-7.41 (m, 1H), 7.30 (t, J=9.0 Hz, 1H), 7.00 (d, J=9.0 Hz,1H), 6.80-6.76 (m, 1H), (dt, J=5.5, 16.0 Hz, 1H), 6.23 (d, J=6.0 Hz,1H), 6.00 (s, 1H), 3.77 (s, 3H), 3.09 (d, J=5.5 Hz, 2H), 2.93-2.87 (m,2H), 2.29 (t, J=6.5 Hz, 2H), 2.13 (s, 3H), 1.42-1.32 (m, 13H). Mass: m/z628 (M⁺+1).

Step-3

TFA (0.5 mL) was added to a solution of 5 in dry DCM (1 mL). Afterstirring 2 h at room temperature, the mixture was concentrated underreduced pressure to give 6. The residue was used in the next stepwithout purification.

Step-4

The residue 6 from Step-3 was diluted in dry DMF (1 mL), and HOBt (10.2mg), EDCI (14.5 mg), N-biotinyl-NH-PEG₂-COOH.DIPEA (52.1 mg), andN-methyl morpholine (23 uL) were added. The resulting mixture wasstirred overnight at room temperature. The crude product was directlyinjected to semi-prep HPLC (TFA modifier) and gave a white solid as aTFA salt. ¹H NMR (DMSO-d₆) 8 ppm: 10.2 (s, 1H), 9.66 (s, 1H), 9.60 (br,1H), 9.08 (br, 1H), 8.25 (s, 1H), 7.78 (m, 3H), 7.68 (dd, J=5.5 & 11.4Hz, 2H), 7.43 (dd, J=2.3 & 8.7 Hz, 1H), 7.30 (m, 2H), 7.00 (d, J=9.2 Hz,1H), 6.64 (m, 1H), 6.36 (d, J=15.1 Hz, 1H), 6.28 (m, 1H), 5.93 (s, 1H),4.21 (dd, J=4.6 & 7.8 Hz, 1H), 4.03 (dd, J=4.6 & 7.8 Hz, 1H), 3.89 (m,1H), 3.71 (s, 3H), 3.38 (m, 9H), 3.28 (t, J=6.4 Hz, 4H), 2.97 (m, 9H),2.71 (dd, J=5.0 & 12.4 Hz, 1H), 2.66 (d, J=4.1 Hz, 3H), 2.47 (d, J=12.4Hz, 1H), 1.95 (m, 6H), 1.1-1.6 (m, 13H); LCMS: m/e 1070.4 (M+1).

tert-Butyl 4-(methylamino)butylcarbamate (4) was prepared by the schemeshown below.

Step-1

To a stirred solution of 4-aminobutanol (5 g, 56.0 mmol) in DCM (100 mL)was added Boc anhydride (12.2 g, 56.0 mmol) and TEA (7.7 mL, 56.0 mmol)at 0° C. and the reaction mixture was stirred at RT for 8 h. After thecompletion of reaction (monitored by TLC), the reaction mixture wasdiluted with water and extracted with DCM (3×25 mL). The organic layerswere dried over anhydrous Na₂SO₄ and concentrated under vacuo. The crudecompound was purified by column chromatography to afford tert-butyl4-hydroxybutylcarbamate (6.0 g, 56.6%) as colorless viscous liquid. ¹HNMR (CDCl₃, 200 MHz): δ 4.63 (bs, 1H), 3.72-3.60 (m, 2H), 3.23-3.08 (m,2H), 1.99-1.50 (m, 4H), 1.43 (s, 9H).

Step-2

To a stirred solution of tert-butyl 4-hydroxybutylcarbamate (7 g, 37.0mmol) in DCM (100 mL) was added TEA (9.35 mg, 92.5 mmol) followed bymethanesulfonyl chloride (5.09 g, 44.4 mmol) over a period of 20 minutesat 0° C. The reaction mixture was further stirred for 18 h at RT. Afterthe completion of reaction (monitored by TLC), the reaction mixture wasdiluted with water (100 mL) and extracted with DCM (3×30 mL). Theorganic layers were dried over anhydrous Na₂SO₄ and concentrated undervacuo. The crude compound was purified by column chromatography toafford (4-tert-butyloxycarbonylamino)-butyl methanesulfonate (5 g,52.2%) as light yellow viscous liquid. ¹H NMR (CDCl₃, 200 MHz): δ 4.62(bs, 2H), 4.25 (t, J=6.0 Hz, 2H), 3.14 (q, J=6.6 Hz, 2H), 3.01 (s, 3H),1.85-1.70 (m, 2H), 1.78-1.54 (m, 2H), 1.44 (s, 9H). Mass: m/z 168(M⁺+1-Boc).

Step-3

To a stirred solution of (4-tert-butyloxycarbonylamino)-butylmethanesulfonate (5 g, 18.7 mmol) in ethanol (25 mL) was addedmethylamine (25 mL) at 0° C. The reaction mixture was stirred for 18 hat RT. After the completion of reaction (monitored by TLC), thevolatiles were removed under reduced pressure to afford tert-butyl4-(methylamino)butylcarbamate (4) (3.4 g, 89.9%) as colorless viscousliquid. The crude compound was used in the next step without furtherpurification. ¹H NMR (CDCl₃, 200 MHz): δ 5.15 (bs, 2H), 3.22-3.10 (m,2H), 2.95 (t, J=7.0 Hz, 2H), 2.70 (s, 3H), 1.90-1.70 (m, 2H), 1.88-1.50(m, 2H), 1.43 (s, 9H).

Example 46 Preparation ofN¹-(4-(methyl((E)-4-oxo-4-(6-(6-(phenylamino)pyrimidin-4-ylamino)pyridin-2-ylamino)but-2-enyl)amino)butyl)-N⁵-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide(I-116)

The title compound can be prepared according to the schemes, steps, andintermediates described in Example 45 by usingN⁴-(6-aminopyridin-2-yl)-N⁶-phenylpyrimidine-4,6-diamine in place of 1in Step-1. The synthesis ofN⁴-(6-aminopyridin-2-yl)-N⁶-phenylpyrimidine-4,6-diamine is described inExample 14, and N⁴-(6-aminopyridin-2-yl)-N⁶-phenylpyrimidine-4,6-diamineis described as an intermediate in the preparation of I-73 in Example15.

Example 47 Preparation ofN¹-(4-(((E)-4-(3-(6-(3-chloro-4-fluorophenoxy)pyrimidin-4-ylamino)phenylamino)-4-oxobut-2-enyl)(methyl)amino)butyl)-N-5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide(I-117)

The title compound can be prepared according to the schemes, steps, andintermediates described in Example 45 by usingN¹-(6-(3-chloro-4-fluorophenoxy)pyrimidin-4-yl)benzene-1,3-diamine inplace of 1 in Step-1. The synthesis ofN¹-(6-(3-chloro-4-fluorophenoxy)pyrimidin-4-yl)benzene-1,3-diamine isdescribed in Example 34 in the preparation of 1-92.

Example 48 Preparation ofN¹-(4-(((E)-4-(3-(6-(3-bromophenylamino)pyrimidin-4-ylamino)phenylamino)-4-oxobut-2-enyl)(methyl)amino)butyl)-N-5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide(I-120)

The title compound can be prepared according to the schemes, steps, andintermediates described in Example 45 by using3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]-phenylamine in place of 1in Step-1. The synthesis of3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]-phenylamine is describedin Example 1, and3-[6-(3-bromophenylamino)-pyrimidin-4-ylamino]-phenylamine is describedas an intermediate in the preparation of 1-1 in Example 3.

Described below are assays used to measure the biological activity ofprovided compounds as inhibitors of ErbB1 (EGFR), ErbB2, ErbB4, TEC,BTK, ITK, BMX, and JAK3.

Example 49 Cloning, Expression and Purification of EGFR-WT and EGFRC797S Mutant Using Baculovirus and Insect Cells (i) Subcloning ofEGFR-WT and Mutant Kinase Domains

Amino acids 696 to 1022 of the EGFR-WT kinase domain (NM_(—)005228,NP_(—)005219.2) was subcloned into the NcoI and HindIII sites of thepFastHTa vector (Invitrogen, Carlsbad, Calif.). To make the EGFR-mutantprotein, the cysteine at position 797 was changed to a serine using theStratagene QuikChange kit (Stratagene, Cedar Creek, Tex.), according tomanufacturer's instructions.

(ii) Expression

P1 baculovirus stocks were generated in SF9 cells via Blue Sky Biotech'ssuspension transfection protocol (Worcester, Mass.). Expression analysiswas conducted in 125 ml culture of SF21 insect cells ((grown in SF900ISFM (Invitrogen cat #10902-088), supplemented with 10 mg/L gentamicin(Invitrogen, Carlsbad, Calif., cat#15710-064)) using a viral load of 0.1ml of virus per 100 ml of cell suspension. Expression was optimizedusing Blue Sky Biotech's Infection Kinetics Monitoring system(Worcester, Mass.).

(iii) Purification

Infected insect cells were pelleted. Cell pellets were resuspended inBlue Sky Biotech's lysis buffer (Worcester, Mass., 1×WX; solubilizationbuffer, containing a protease inhibitor cocktail of leupeptin,pepstatin, PMSF, aprotinin and EDTA) at a ratio of 10 ml per gram of wetcell paste. Cells were lysed by sonication and the lysate was clarifiedby centrifugation at 9,000RPM for 30 minutes in a GSA rotor. 500 μl bedvolume of NiNTA resin (Qiagen, Valencia, Calif.) was added to thesupernatants and batch bound for two hours with constant agitation. Thematerial was transferred by gravity into an empty 2 ml column. Thecolumn was washed with 2 ml of wash buffer (Blue Sky Biotech, Worcester,Mass., 1×WX, 25 mM imidazole). The protein was eluted with1×WX+imidazole at varying concentrations: Elution 1: 75 mM imidazole (2fractions, 1 column volume); Elution 2: 150 mM imidazole (2 fractions, 1column volume); Elution 3: 300 mM imidazole (2 fractions, 1 columnvolume). All the elution fractions were analyzed by SDS page followed byCoomassie staining and Western Blotting using anti-penta-his antibody(Qiagen, Valencia, Calif.). The carboxy-terminal six-histidine “tag” wasremoved from some of the purified protein using AcTEV Protease kit(Invitrogen, Carlsbad, Calif., Cat#12575-015), following manufacturer'sinstructions. All the samples (pre- and post-Tev cut) were analyzed bySDS page followed by Coomassie staining and Western Blotting, asdescribed above.

Example 50 Mass Spectrometry for EGFR

EGFR wild type and EGFR mutant (C797S) was incubated with 10-fold excessof compound I-1 for 1 hr and 3 hrs. 1 ul aliquots of the samples (totalvolume 5-8 ul) were diluted with 10 ul of 0.1% TFA prior to micro C4ZipTipping directly onto the MALDI target using Sinapinic acid as thedesorption matrix (10 mg/ml in 0.1% TFA:Acetonitrile 50:50). Intact massmeasurement reveals that the wild type has a nominal mass of about 37557and the mutant slightly lower at 37500. Reactivity was only observed forthe wild type EGFR with a new peak appearing at a mass consistent with asingle site covalent modification with compound I-1 which has a mass of410 Da. (See FIG. 8). The mutant EGFR(C797S) showed no significantreactivity even after 3 hrs, confirming modification of the cysteine ofinterest, Cys797.

Example 51 Omnia Assay Protocol for Potency Assessment Against EGFR (WT)and EGFR (T790M/L858R) Active Enzymes

The protocol below describes continuous-read kinase assays to measureinherent potency of compounds against active forms of EGFR (WT) and EGFR(T790M/L858R) enzymes. The mechanics of the assay platform are bestdescribed by the vendor (Invitrogen, Carlsbad, Calif.) on their websiteat the following URL: http://www.invitrogen.com/content.cfm?pageid=1338orhttp://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Drug-Discovery/Target-and-Lead-Identification-and-Validation/KinaseBiology/KB-Misc/Biochemical-Assays/Omnia-Kinase-Assays.html.

Briefly, 10× stocks of EGFR-WT (PV3872) from Invitrogen andEGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif.,1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates(KCZ1001) were prepared in IX kinase reaction buffer consisting of 20 mMTris, pH 7.5, 5 mM MgCl₂, 1 mM EGTA, 5 mM β-glycerophosphate, 5%glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of eachenzyme were pre-incubated in a Corning (#3574) 384-well, white,non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27°C. with a 0.5 μL volume of 50% DMSO and serially diluted compoundsprepared in 50% DMSO. Kinase reactions were started with the addition of45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90seconds for 60 minutes at λ_(ex)360/λ_(em)485 in a Synergy⁴ plate readerfrom BioTek (Winooski, Vt.). At the conclusion of each assay, progresscurves from each well were examined for linear reaction kinetics and fitstatistics (R², 95% confidence interval, absolute sum of squares).Initial velocity (0 minutes to ˜30 minutes) from each reaction wasdetermined from the slope of a plot of relative fluorescence units vstime (minutes) and then plotted against inhibitor concentration toestimate IC₅₀ from log [Inhibitor] vs Response, Variable Slope model inGraphPad Prism from GraphPad Software (San Diego, Calif.).

The EGFR-WT- and EGFR T790M/L858R-modified optimized reagent conditionsare:

-   -   [EGFR-WT]=5 nM, [ATP]=15 mM, [Y12-Sox]=5 mM (ATP KMapp˜12 mM);        and    -   [EGFR-T790M/L858R]=3 nM, [ATP]=50 mM, [Y12-Sox]=5 mM (ATP        KMapp˜45 mM).

Example 52

Table 7 shows the activity of selected compounds of this invention inthe EGFR inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≦10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≧10,000 nM.

TABLE 7 EGFR Wild Type and EGFR (mutant C797S) Inhibition Data EGFR EGFR(T790M/L858R) Compound # inhibtion inhibiton I-1 A A I-2 A A I-3 A A I-4A B I-5 A A I-6 B B I-7 A B I-8 A D I-9 A C I-10 A C I-11 B E I-12 B EI-13 A B I-14 A B I-15 A B I-16 A B I-17 A B I-18 A A I-19 A A I-46 A AI-47 A E I-48 C C I-49 A A I-50) A B I-51 A D I-52 A A I-53 B C I-54 A AI-55 A A I-56 A A I-57 — C I-58 — C I-59 A A I-60 D C I-61 — A I-63 A AI-65 A B I-66 A C I-67 A A I-68 A — I-69 B — I-70 A — I-71 A — I-73 A —I-75 A — I-78 A — I-79 A — I-80 C — I-81 A — I-82 A — I-83 A — I-84 B —I-85 B — I-86 B — I-87 B — I-88 A — I-89 A — I-90 A — I-91 A — I-92) A —I-93 A — I-94 A A I-95 A — I-97 A — I-98 A — I-99 A — I-100 A — I-101 A— I-102 A — I-103 A — I-104 A — I-105 A —

Example 53 Cellular Assays for EGFR Activity

Compounds were assayed in A431 human epidermoid carcinoma cells using amethod substantially similar to that described in Fry, et al., Proc.Natl. Acad. Sci. USA Vol 95, pp 12022-12027, 1998. Specifically, A431human epidermoid carcinoma cells were grown in 6-well plates to 90%confluence and then incubated in serum-free media for 18 hr. Duplicatesets of cells were treated with 1 μM designated compound for 2, 5, 10,30, or 60 min. Cells were washed free of the compound with warmedserum-free medium, incubated for 2 hr, washed again, incubated another 2hr, washed again, and then incubated another 2 hr washed again andincubated for additional 2 hr and then stimulated with 100 ng/ml EGF for5 min. Extracts were made as described Fry, et al. FIG. 1 depicts theEGFR inhibiting activity of compound I-1.

Compounds were assayed in A431 human epidermoid carcinoma cells using amethod substantially similar to that described in Fry, et al.Specifically, A431 human epidermoid carcinoma cells were grown in 6-wellplates to 90% confluence and then incubated in serum-free media for 18hr. Cells were then treated with 10, 1, 0.1, 0.01, or 0.001 μM testcompound for 1 hr. Cells were then stimulated with 100 ng/ml EGF for 5min, and extracts were made as described in Fry, et al. 20 ug totalprotein from lysates were loaded on gel and blots were probed for eitherEGFR phosphorylation or p42/p44 Erk phosphorylation.

Dose response inhibition of EGFR phosphorylation and p42/p44 Erkphosphorylation with compound I-16 and I-17 in A431 cells is depicted inFIG. 3. Dose response inhibition of EGFR phosphorylation and p42/p44 Erkphosphorylation with compound I-19 in A431 cells is depicted in FIG. 4.Dose response inhibition of EGFR phosphorylation with compound I-1 inA431 cells, as compared with its “reversible control” compound(I^(R)-3), is depicted in FIG. 5.

Example 54 Washout Experiment for EGFR Activity

A431 human epidermoid carcinoma cells were grown in 6-well plates to 90%confluence and then incubated in serum-free media for 18 hr. Duplicatesets of cells were treated with 1 μM designated compound for 1 hr. Oneset of cells was then stimulated with 100 ng/ml EGF for 5 min, andextracts were made as described. The other set of cells was washed freeof the compound with warmed compound-free medium, incubated for 2 hr,washed again, incubated another 2 hr, washed again, and then incubatedanother 2 hr washed again and incubated for additional 2 hr and thenstimulated with EGF. The results of this experiment are depicted in FIG.6 where it is shown that compound I-1 maintains enzyme inhibition after“washout” whereas its “reversible control” compound (I^(R)-3) was washedaway in the experiment thereby resulting in reactivated enzyme activity.

Example 55 Mass Spectrometry for ErbB4

ErbB4 kinase domain (Upstate) was incubated with compound for 90 minutesat 10-fold excess of compound I-1 to protein. 1 ul aliquots of thesamples (total volume of 4.24 ul) were diluted with 10 ul of 0.1% TFAprior to micro C4 ZipTipping directly onto the MALDI target usingSinapinic acid as the desorption matrix (10 mg/ml in 0.1%TFA:Acetonitrile 50:50). For intact protein mass measurement theinstrument was set in Linear mode using a pulsed extraction setting of16,952 for the myoglobin standard used to calibrate the instrument(Shimadzu Axima TOF²).

Intact ErbB4 protein occurs at MH+ of 35850 with corresponding sinapinic(matrix) adducts occurring about 200 Da higher. A stoichiometricincorporation of the compound I-1 (Mw of 410 Da) produced a new masspeak which is approximately 410 Da higher (MH+ of 36260). This isconsistent with covalent modification of ErbB4 with compound I-1 asdepicted in FIG. 7.

Example 56 ErbB1, ErbB2 and/or ErbB4 Kinase Inhibition

Compounds of the present invention were assayed as inhibitors of one ormore of ErbB1, ErbB2, and/or ErbB4 in a manner substantially similar tothe method described by Invitrogen Corp (Invitrogen Corporation, 1600Faraday Avenue, Carlsbad, Calif., CA;http://www.invitrogen.com/downloads/Z-LYTE_Brochure_(—)1205.pdf) usingthe Z′-LYTE™ biochemical assay procedure or similar biochemical assay.The Z′-LYTE™ biochemical assay employs a fluorescence-based,coupled-enzyme format and is based on the differential sensitivity ofphosphorylated and non-phosphorylated peptides to proteolytic cleavage.

Example 57

Table 8 shows the activity of selected compounds of this invention inthe ErbB inhibition assay. The compound numbers correspond to thecompound numbers in Table 5.

TABLE 8 ErbB1, ErbB2, and/or ErbB4 Inhibition Data ErbB1 ErbB2 ErbB4Compound # % Inhibition % Inhibition % Inhibition I-1  99% @ 10 nM 76% @1 μM  86% @ 1 μM 61% @ 100 nM  64% @ 100 nM I-2  98% @ 100 nM 96% @ 1 μM 75% @ 1 μM  75% @ 10 nM 39% @ 100 nM I-3  96% @ 100 nM 89% @ 1 μM  95%@ 1 μM  56% @ 10 nM I-4 100% @ 10 nM 86% @ 1 μM  78% @ 1 μM I-5 100% @10 nM 86% @ 1 μM  95% @ 1 μM I-6 100% @ 100 nM 84% @ 1 μM  97% @ 1 μMV49% @ 10 nM I-7 100% @ 100 nM 89% @ 1 μM 100% @ 1 μM  53% @ 10 nM I-8 83% @ 10 nM —  57% @ 1 μM I-9 100% @ 1 μM —  75% @ 1 μM I-10  96% @ 1μM — — I-11  79% @ 1 μM — — I-12  82% @ 1 μM — — I-13  92% @ 1 μM 98% @1 μM — I-14  96% @ 1 μM — — I-15  98% @ 1 μM — — I-16  98% @ 1 μM 87% @100 nM  93% @ 100 nM 24% @ 10 nM  26% @ 10 nM I-17  95% @ 1 μM 89% @ 1μM  94% @ 1 μM I-18 — 91% @ 1 μM  94% @ 1 μM I-19  96% @ 1 μM 98% @ 1 μM 97% @ 1 μM

Example 58 Mass Spectrometry for TEC Kinase

TEC kinase (45 pmols; Invitrogen) was incubated with (I-13) (450 pmols)for 3 hrs at 10× access prior to tryptic digestion. Iodoacetamide wasused as the alkylating agent after compound incubation. A control sample(45 pmols) was also prepared which did not have the addition of (I-13).For tryptic digests a 5 ul aliquot (7.5 pmols) was diluted with 15 ul of0.1% TFA prior to micro C18 Zip Tipping directly onto the MALDI targetusing alpha cyano-4-hydroxy cinnamic acid as the matrix (5 mg/ml in 0.1%TFA:Acetonitrile 50:50).

As depicted in FIG. 11, the expected peptide (GCLLNFLR) to be modifiedwas immediately evident at MH+ of 1358.65. This is the mass to beexpected when compound I-13, with an adduct mass of 423.17, is added tothe peptide mass of 935.51. The peptide was also quite evident in thecontrol sample as modified by Iodacetamide at MH+ of 992.56.Interestingly the Iodoacetamide modified peptide was not evident in thedigest reacted with compound I-13 indicating that the reaction wascomplete. There was no evidence of any other modified peptides.

Evidence of compound I-13 was observed at MH+ of 424.20 in the low massrange of the spectra. The fragmentation spectra of the 424.20 peak didshow many diagnostic fragments that were apparent in the PSD spectra ofthe modified peptide at 1358.65 (see FIG. 11).

To further verify the presence of the modified peptide with compoundI-13, the peptide at MH+ of 1358.65 was subjected to PSD (MS/MS)analsysis. A correlational analysis with the homosapien databaseidentified the correct peptide modified by 1-13.

Instrumental:

For tryptic digests the instrument was set in Reflectron mode with apulsed extraction setting of 2200. Calibration was done using the LaserBiolabs Pep Mix standard (1046.54, 1296.69, 1672.92, 2093.09, 2465.20).For CID/PSD analysis the peptide was selected using cursors to set iongate timing and fragmentation occurred at a laser power about 20% higherand He was used as the collision gas for CID. Calibration for fragmentswas done using the P14R fragmentation calibration for the Curved fieldReflectron.

Example 59 Omnia Assay Protocol for Potency Assessment Against BTK

The Omnia Assay Protocol for potency assessment against BTK is performedin a substantially similar manner as that described in Example 25 aboveexcept that the modified BTK-optimized reagent conditions are:

-   -   [BTK]=5 nM, [ATP]=40 mM, [Y5-Sox]=10 mM (ATP KMapp˜36 mM).

Example 60

Table 9 shows the activity of selected compounds of this invention inthe BTK inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≦10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≧10,000 nM.

TABLE 9 BTK Inhibition Data Compound # BTK Inhibition I-1 B I-2 B I-3 BI-4 A I-5 A I-6 C I-7 B I-8 E I-9 C I-10 C I-11 E I-12 E I-13 A I-14 CI-15 B I-16 B I-17 A I-18 B I-19 B I-46 C I-47 E I-48 B I-49 A I-50 DI-51 E I-52 A I-53 B I-54 C I-55 B I-56 B I-57 D I-58 D I-59 C I-60 DI-61 B I-62 A I-63 A I-64 A I-65 A I-66 A I-67 B I-68 B I-69 C I-70 CI-71 D I-73 A I-75 A I-78 A I-79 A I-80 D I-81 A I-82 A I-83 A I-84 DI-85 B I-86 B I-87 E I-88 A I-89 A I-90 A I-91 A I-92 B I-93 D I-94 AI-95 A I-97 C I-98 C I-99 A I-100 A I-101 A I-102 B I-103 C I-104 AI-105 B

Example 61 BTK Ramos Cellular Assay

Compounds I-13 and (I-52) were assayed in Ramos human Burkitt lymphomacells. Ramos cells were grown in suspension in T225 flasks, spun down,resuspended in 50 mls serum-free media and incubated for 1 hour.Compound was added to Ramos cells in serum free media to a finalconcentration of 1, 0.1, 0.01, or 0.001 μM. Ramos cells were incubatedwith compound for 1 hour, washed again and resuspended in 100 μlserum-free media. Cells were then stimulated with 1 μg of goat F(ab′)2Anti-Human IgM and incubated on ice for 10 minutes to activate B cellreceptor signaling pathways. After 10 minutes, the cells were washedonce with PBS and then lysed on ice with Invitrogen Cell Extractionbuffer. 16 μg total protein from lysates was loaded on gels and blotswere probed for phosphorylation of the BTK substrate PLCγ2. At 1 μM 1-13showed 85% inhibition and (I-52) showed 50% inhibition of BTK signalingin Ramos cells. Additional dose response inhibition of BTK signalingwith 1-13 is depicted in FIG. 9.

Table 10 provides inhibition data for selected compounds in Ramos cells.The compound numbers correspond to the compound numbers in Table 5.Compounds having an activity designated as “A” provided an IC₅₀≦10 nM;compounds having an activity designated as “B” provided an IC₅₀ of10-100 nM; compounds having an activity designated as “C” provided anIC₅₀ of 100-1000 nM; compounds having an activity designated as “D”provided an IC₅₀ of ≧1000 nM.

TABLE 10 BTK Ramos Cellular Inhibition Data Compound # BTK Inhibition(nM) I-5 C I-13 B I-17 D I-63 A I-64 B I-65) A I-66) A I-78 A I-79 AI-81 A I-82 A I-95 A I-99 A I-100 B

Example 62 Washout Experiment with Ramos Cells for BTK Activity

Ramos cells were serum starved for one hour in RPMI media+1% glutamineat 37° C. After starvation, Ramos cells were treated with 100 nMcompound diluted in serum free RPMI media for 1 hour. After compoundtreatment, the media was removed and cells were washed withcompound-free media. Subsequently, Ramos cells were washed every 2.hours and resuspended in fresh compound-free media. Cells were collectedat specified timepoints, treated with 1 ug anti-human IgM (SouthernBiotech cat #2022-01) for 10 minutes on ice to induce BCR signaling andthen washed in PBS. Ramos cells were then lysed in Cell ExtractionBuffer (Invitrogen FNN0011) supplemented with Roche complete proteaseinhibitor tablets (Roche 11697498001) and phosphatase inhibitors (Roche04 906 837 001) and 18 ug total protein lysate was loaded in each lane.Inhibition of BTK kinase activity was assayed by measuring its substrate(PLCγ2) phosphorylation by western blot with phospho-specific antibodiesfrom Cell Signaling Technologies cat#3871. FIG. 10 depicts the resultsof compound I-13 in the washout experiment at 0 hour, 4 hour, 6 hour,and 8 hour time points. As shown in FIG. 10, compound I-13 maintainsinhibition of BTK for 8 hours.

Table 11 provides data for selected compounds in the Ramos washoutassay.

TABLE 11 BTK Washout Data Compound # BTK Inhibition Type I-13irreversible I-63 irreversible I-64 irreversible I-65 reversible I-66irreversible I-82 irreversible

Example 63 Mass Spectrometry for BTK

Intact BTK was incubated for 1 hr at a 10× fold excess of compound I-63or I-66 to protein. Aliquots (2 ul) of the samples were diluted with 10ul of 0.1% TFA prior to micro C4 ZipTipping directly onto the MALDItarget using Sinapinic acid as the desorption matrix (10 mg/ml in 0.1%TFA:Acetonitrile 20:80). Mass spectrometry traces are shown in FIG. 12and FIG. 13. The top panels of FIGS. 12 and 13 show the mass spec traceof the intact BTK protein (m/z 81,169 Da). The bottom panels show themass spec trace when BTK was incubated with compound I-63 (mw 424.5) orcompound I-66 (mw 425.5) in FIGS. 12 and 13, respectively. The centroidmass (m/z 81,593 kDa) in the bottom panel of FIG. 12 shows a positiveshift of about 424 Da, indicating complete modification of BTK bycompound I-63. The centroid mass (m/z 81,593 kDa) in the bottom panel ofFIG. 13 shows a positive shift of about 407 Da, indicating completemodification of BTK by compound I-66.

Example 64 Omnia Assay Protocol for Potency Assessment Against ActiveForms of ITK Kinase

This example describes continuous-read kinase assays to measure inherentpotency of compound against active forms of ITK enzymes as described inExample 10 above except that the modified ITK-optimized reagentconditions are:

-   -   [ITK]=10 nM, [ATP]=25 μM, [Y6-Sox]=10 μM (ATP K_(Mapp)=33 μM).

Example 65

Table 12 shows the activity of selected compounds of this invention inthe ITK inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≦10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≧10,000 nM.

TABLE 12 ITK Inhibition Data Compound # ITK inhibition I-10 E I-13 DI-14 D I-15 D I-63 D I-64 B I-65 B I-66 D I-78 B I-79 A I-81 B I-88 BI-89 C I-90 C I-94 B I-95 E I-98 E I-100 E I-101 C

Example 66 Omnia Assay Protocol for Potency Assessment Against ActiveForms of BMX Kinase

This example describes continuous-read kinase assays to measure inherentpotency of compound against active forms of BMX enzymes as described inExample 10 above except that the modified BMX-optimized reagentconditions are:

-   -   [BMX]=2.5 nM, [ATP]=100 μM, [Y5-Sox]=7.5 μM (ATP K_(Mapp)=107        μM).

Example 67

Table 13 shows the activity of selected compounds of this invention inthe BMX inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≦10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≧10,000 nM.

TABLE 13 BMX Inhibition Data Compound # BMX inhibition I-10 — I-13 AI-14 — I-15 — I-63 A I-64 B I-65 A I-66 A I-78 B I-79 A I-81 A I-94 A

Example 68 Omnia Assay Protocol for Potency Assessment Against theActive Form of Janus-3 Kinase (JAK3)

The Omnia Assay Protocol for potency assessment against JAK3 isperformed in a substantially similar manner as that described in Example25 above except that the modified JAK3-optimized reagent conditions are:

-   -   [JAK3]=5 nM, [ATP]=5 μM, [Y12-Sox]=5 μM (ATP KMapp ˜5 μM).

Example 69

Table 14 shows the activity of selected compounds of this invention inthe JAK3 inhibition assay. The compound numbers correspond to thecompound numbers in Table 5. Compounds having an activity designated as“A” provided an IC₅₀≦10 nM; compounds having an activity designated as“B” provided an IC₅₀ 10-100 nM; compounds having an activity designatedas “C” provided an IC₅₀ of 100-1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀ of 1000-10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≧210,000 nM.

TABLE 14 JAK3 Inhibition Data Compound # JAK3 Inhibition I-1  A I-2  AI-3  A I-4  A I-5  A I-6  D I-7  C I-8  D I-9  C I-10 C I-11 E I-12 EI-13 C I-14 A I-15 B I-16 B I-17 B I-18 B I-19 C I-46 D I-47 E I-48 EI-49 A I-50 C I-51 E I-52 A I-53 C I-54 D I-55 B I-56 B I-57 E I-58 CI-59 D I-60 D I-61 B I-62 C I-63 B I-64 A I-65 A I-66 C I-67 A I-68 AI-69 C I-70 C I-71 D I-73 A I-75 C I-78 A I-79 A I-80 E I-81 A I-82 BI-84 E I-85 D I-86 E I-92 B I-93 E I-94 A

Example 70 Measuring ErbB occupancy

Protein samples from ErbB expressing cells (e.g., A431 human epidermoidcarcinoma lysates or SKOV3 ovarian cancer tumor cells grown in vivo)that are or are not previously treated with a covalent inhibitor will beincubated with a covalent probe compound for 1 h. The target-covalentprobe complex will be captured using streptavidin beads or using anantibody that recognizes the ErbB target. The captured proteins will beseparated by SDS-PAGE and analyzed by Western blot using thecomplementary approach, i.e. an anti-ErbB antibody for the complexescaptured with strepatavidin or streptavidin for the complexes capturedusing the anti-ErbB antibody. The amount of target that is bound to thecovalent probe will be quantitated and compared to the amount of totalErbB protein to determine the percentage of target that is occupied bythe covalent drug.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

1-4. (canceled)
 5. A method for inhibiting one or more of ErbB1, ErbB2,ErbB3, or ErbB4, or a mutant thereof, activity in a patient or in abiological sample comprising the step of administering to said patientor contacting said biological sample with a compound selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.
 6. The method accordingto claim 5, wherein the one or more of ErbB1, ErbB2, or ErbB4, or amutant thereof, activity is inhibited irreversibly.
 7. The methodaccording to claim 5, wherein the one or more of ErbB1, ErbB2, or ErbB4,or a mutant thereof, activity is inhibited irreversibly by covalentlymodifying Cys797 of ErbB1, Cys805 of ErbB2 or Cys803 of ErbB4.
 8. Amethod for treating an ErbB1-, ErbB2-, ErbB3-, or ErbB4-mediateddisorder in a patient in need thereof, comprising the step ofadministering to said patient a compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.
 9. The method accordingto claim 8, wherein the disorder is a carcinoma selected from breastcancer, glioblastoma, lung cancer, cancer of the head and neck,colorectal cancer, bladder cancer, non-small cell lung cancer, squamouscell carcinoma, salivary gland carcinoma, ovarian carcinoma, orpancreatic cancer.
 10. The method according to claim 8, wherein thedisorder is selected from neurofibromatosis type I (NF1),neurofibromatosis type II (NF2) Schwann cell neoplasms (e.g. MPNST's),or a Schwannoma. 11-39. (canceled)